Combination tumor treatment with drug-loaded, bispecific ligand-targeted minicells and interferon-gamma

ABSTRACT

Compositions and methods are provided for cancer treatments. The methodology entails, for instance, administering to a cancer patient a first composition comprising a plurality of bacterially derived intact minicells or intact killed bacterial cells, each of which encompasses an anti-neoplastic agent and carries a bispecific ligand on the surface, the ligand having specificity for a mammalian cell component, and a second composition comprising interferon-gamma (IFN-gamma) or an agent that increases the expression of IFN-gamma in the subject. The compositions include the first composition and the second composition as described, optionally with additional anti-neoplastic agents.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. patent application Ser. No.14/506,138, filed on Oct. 3, 2014, which claims priority from U.S.Provisional Patent Application No. 61/887,258, filed on Oct. 4, 2013.The contents of these application are incorporated herein by referencein their entirety.

BACKGROUND

Currently, most drugs used for treating cancer are administeredsystemically. Although systemic delivery of cytotoxic anticancer drugsplays a crucial role in cancer therapeutics, it also engenders seriousproblems. For instance, systemic exposure of normal tissues/organs tothe administered drug can cause severe toxicity. This is exacerbated bythe fact that systemically delivered cancer chemotherapy drugs oftenmust be delivered at very high dosages to overcome poor bioavailabilityof the drugs and the large volume of distribution within a patient.Also, systemic drug administration can be invasive, as it often requiresthe use of a secured catheter in a major blood vessel. Because systemicdrug administration often requires the use of veins, either peripheralor central, it can cause local complications such as phlebitis.Extravasation of a drug also can lead to vesicant/tissue damage at thelocal site of administration, such as is commonly seen uponadministration of vinca alkaloids and anthracyclines.

Another challenge in cancer therapy is evasion by tumor cells fromimmune surveillance. Interactions between the immune system andmalignant cells play an important role in tumorigenesis. Failure of theimmune system to detect and reject transformed cells may lead to cancerdevelopment. Tumors use multiple mechanisms to escape fromimmune-mediated rejection. Many of these mechanisms are now known on acellular and molecular level. Despite this knowledge, cancerimmunotherapy is still not an established treatment in the clinic.

SUMMARY

The present inventors discovered that an animal undergoing cancertherapy with anti-neoplastic drug-loaded, bispecific antibody-targeted,minicells exhibits a greater anti-tumor response to the drug when theanimal is suffering from a concomitant viral infection. Furtherinvestigation revealed that the observed enhancement in the therapeuticefficacy of an anti-cancer drug in this context arose from synergismbetween the tumor-killing capability of the administered, drug-loaded,bispecific antibody-targeted minicells and an activated host-immuneresponse against tumor cells, itself due to increased expression ofinterferon-gamma (IFN-gamma or IFNγ) that the viral infection triggered.

IFN-gamma itself has been investigated for its potential anti-neoplasticuse, both in monotherapy and in combination with other anti-neoplasticagents. Such investigations have not led to clinical success, however.For instance, the combination treatment of IFN-alpha and IFN-gammafailed to exhibit an improvement over treatment with IFN-alpha alone.See, e.g., Kloke et al., Eur. J. Haematol. 48: 93-8 (1992), and Wandl etal., Semin. Oncol. 19: 88-94 (1992). The only IFN-gamma indicationsapproved by the U.S. Food and Drug Administration (FDA) are for treatingchronic granulomatous disease (CGD) and severe malignant osteopetrosis(bone disease).

In one of its aspects, therefore, the present disclosure provides amethod for treating a tumor in a subject. The method entailsadministering to the subject (A) a first composition comprising aplurality of bacterially derived intact minicells and/or killedbacterial cells, each of which minicells and killed cells encompasses ananti-neoplastic agent and are targeted to a tumor cell surface receptorvia a ligand attached to the minicell surface, and (B) a secondcomposition comprising IFN-gamma or an agent that increases theexpression or activity of IFN-gamma in the subject.

In some aspects, the second composition comprises IFN-gamma protein, inparticular a pharmaceutically suitably purified IFN-gamma protein. Insome aspects, the second composition comprises a viral vaccine. In someaspects, the second composition comprises a nucleic acid encodingIFN-gamma.

In some aspects, the first composition comprises from about 10⁹ to about10¹⁰ minicells or killed bacterial cells.

In some aspects, the anti-neoplastic agent is a radionuclide. In someaspects, the anti-neoplastic agent is a chemotherapy drug. In someaspects, the anti-neoplastic agent is a functional nucleic acid or apolynucleotide encoding a functional nucleic acid. In some aspects, thefunctional nucleic acid inhibits a gene that promotes tumor cellproliferation, angiogenesis or resistance to chemotherapy and/or thatinhibits apoptosis or cell cycle arrest. In some aspects, the functionalnucleic acid is selected from siRNA, miRNA, shRNA, lincRNA, antisenseRNA, or ribozyme.

Also provided are packages, products or kits comprising a firstcomposition comprising a plurality of bacterially derived intactminicells or intact killed bacterial cells, each of which encompasses ananti-neoplastic agent and carries a ligand on the surface wherein theligand has specificity to a non-phagocytic mammalian cell surfacereceptor, and a second composition comprising interferon-gamma(IFN-gamma) or an agent that increases the expression of IFN-gamma inthe subject.

In another embodiment, provided is a composition comprising (a) aplurality of bacterially derived intact minicells or intact killedbacterial cells, each of which encompasses an anti-neoplastic agent andcarries a ligand on the surface wherein the ligand has specificity to anon-phagocytic mammalian cell surface receptor, and (b) IFN-gamma or anagent that increases the expression of IFN-gamma in the subject.

Other objects, features, and advantages are apparent from the followingdescription. The detailed description and specific examples are givenfor illustration only, since various changes and modifications withinthe spirit and scope of the particular embodiments are apparent fromthis description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C present charts of tumor volumes (y axis on the left) andserum IFN-gamma concentrations (y axis on the right), measured atdifferent time points (x axis, shown as number of doses), for three dogsA, B, and C, respectively. These charts show that the response of thetumor to the drug was much greater when serum concentrations ofIFN-gamma were elevated.

FIG. 2 illustrates the effects of combined treatment, with IFN-gamma andbispecific ligand-targeted and doxorubicin-packaged intact minicells, ofhuman alveolar adenocarcinoma tumor xenografts, established in 6week-old female athymic nude mice with a tumor size of about 285 mm³.Group 1 mice received saline, Group 2 mice received IFN-gamma only,Group 3 mice received ^(EGFR)minicells_(Dox), and Group 4 mice received^(EGFR)minicells_(Dox) and IFN-gamma. In this example and those tofollow, the triangles below the x axis indicate the time of dosing.

FIG. 3 depicts the effects of combined treatment, with IFN-gamma andbispecific ligand-targeted and doxorubicin-packaged, intact minicells,of human breast tumor xenografts established in 6 week-old femaleathymic nude mice with a moderate tumor size, about 145 mm³. Group 1mice received saline, Group 2 mice received IFN-gamma only, Group 3 micereceived ^(EGFR)minicells_(Dox), and Group 4 mice received^(EGFR)minicells_(Dox) and IFN-gamma.

FIG. 4 illustrates the effects of combined treatment, with IFN-gamma andbispecific ligand-targeted and doxorubicin-packaged intact minicells, ofhuman breast tumor xenografts established in 6 week-old female athymicnude mice with large tumor size, about 250 mm³. Group 1 mice receivedsaline, Group 2 mice received IFN-gamma only, Group 3 mice received^(EGFR)minicells_(Dox), and Group 4 mice received ^(EGFR)minicells_(Dox)and IFN-gamma.

FIG. 5 depicts the effects of combined treatment, with IFN-gamma andbispecific ligand-targeted and doxorubicin-packaged intact minicells, ofhuman breast tumor xenografts established in 6 week-old female athymicnude mice with a very large tumor size, between about 265 and about 600mm³. All four mice received ^(EGFR)minicells_(Dox) and IFN-gamma.

FIG. 6 portrays the effects of combined treatment, with IFN-gamma (twodifferent doses) and bispecific ligand-targeted and doxorubicin-packagedintact minicells, of human alveolar adenocarcinoma tumor xenograftsestablished in 6 week-old female athymic nude mice with a tumor size ofabout 100 mm³. Group 1 mice received saline, Group 2 mice received^(EGFR)minicells_(Dox), Group 3 mice received ^(EGFR)minicells_(Dox) and0.75×10⁴ IU of IFN-gamma, twice per week, and Group 4 mice received^(EGFR)minicells_(Dox) and 0.5×10⁴ IU of IFN-gamma, three times perweek.

FIG. 7 illustrates 20 subfamilies and 58 members of human receptortyrosine kinases (excerpted from Lemmon and Schlessinger, Cell 141:1117-134 (2010)).

DETAILED DESCRIPTION

As noted above, the inventors determined that administeringanti-neoplastic drug-loaded, bispecific antibody-targeted minicells to apatient with a tumor, in a situation where the patient is exposed to anelevated level of INF-gamma, results in an anti-tumor response that isgreatly improved compared to what is observed when IFN-gamma is notactivated, e.g., when its level is below detection limits. This synergybetween minicell-mediated anti-tumor activity and elevated IFN-gamma isapparent from the magnitude of increased tumor response. Withoutcommitting to any particular mechanism(s), the inventors contemplatethat the approach described here exploits critical pathways in immunestimulation that are important in host anti-tumor responses. Thebacterially derived minicells and IFN-gamma elicit different pathways inimmune stimulation, which collectively is important in augmenting theanti-tumor response that the anti-neoplastic drug initiates uponintracellular delivery to tumor cells via the bispecificantibody-targeted minicells, in accordance with the present disclosure.

The inventors also discovered that blood vessels around tumor cellsdisplay a loss of integrity; that is, the vessels have largefenestrations and are “leaky,” even in the blood brain barrier (BBB)environment. In contravention of conventional understanding, therefore,particles that are as large as minicells, i.e., much larger than theabove-discussed consensus pore size limitations of the BBB, neverthelessare smaller than the fenestrations in the walls of the leaky bloodvessel; hence, they can extravasate passively through thesefenestrations and into the tumor microenvironment.

Upon entering the tumor microenvironment, minicells are able to triggerreceptor-mediated internalization by the host tumor cells and to betaken up by them. Thus, a minicell that is packaged with ananti-neoplastic agent will release the agent into the cytoplasm of thetumor cell, killing it.

Although IFN-gamma has been suggested for use in tumor therapy, itsclinical application has been limited to date, in no small part due toits high toxicity. The ability of IFN-gamma to stimulate immune responseto tumor cells also has not seen much success. In the context of thepresent invention, therefore, the role played by IFN-gamma not only isadvantageous but also is truly surprising.

In one of its aspects, therefore, the present disclosure provides atreatment for a tumor that entails administering to the patient with thetumor a composition comprised of a plurality of intact, bacteriallyderived minicells carrying an anti-neoplastic agent, while alsoadministering to the patient an agent that increases his or her level ofIFN-gamma. According to another aspect, killed bacterial cells can beused with or in lieu of minicells, since such cells likewise can beloaded with anti-cancer drug for release upon uptake into target tumorcells. See, e.g., published international application WO/2008/012695,the contents of which are incorporated here by reference.

The administration of a composition containing drug-loaded minicelland/or killed bacterial cell preferably is systemic, e.g., intravenousor intra-arterial. Further, the IFN-gamma or an agent inducing theexpression of IFN-gamma can be administered by a route that isdifferent, i.e., subcutaneous or intramuscular, but need not be. Theminicell and/or killed bacterial cell therapeutic can be administeredconcomitantly with the IFN-gamma or at different times.

(A) Definitions

Unless defined otherwise, all technical and scientific terms used inthis description have the same meaning as commonly understood by thoseskilled in the relevant art.

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.Other terms and phrases are defined throughout the specification.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

“Cancer,” “neoplasm,” “tumor,” “malignancy” and “carcinoma,” usedinterchangeably herein, refer to cells or tissues that exhibit anaberrant growth phenotype characterized by a significant loss of controlof cell proliferation. The methods and compositions of this disclosureparticularly apply to malignant, pre-metastatic, metastatic, andnon-metastatic cells.

“Drug” refers to any physiologically or pharmacologically activesubstance that produces a local or systemic effect in animals,particularly mammals and humans.

“Individual,” “subject,” “host,” and “patient,” terms usedinterchangeably in this description, refer to any mammalian subject forwhom diagnosis, treatment, or therapy is desired. The individual,subject, host, or patient can be a human or a non-human animal. Thus,suitable subjects can include but are not limited to non-human primates,cattle, horses, dogs, cats, guinea pigs, rabbits, rats, and mice.

The terms “treatment,” “treating,” “treat,” and the like refer toobtaining a desired pharmacological and/or physiologic effect in a tumorpatient. The effect can be prophylactic in terms of completely orpartially preventing tumor or symptom thereof and/or can be therapeuticin terms of a partial or complete stabilization or cure for tumor and/oradverse effect attributable to the tumor. Treatment covers any treatmentof a tumor in a mammal, particularly a human. A desired effect, inparticular, is tumor response, which can be measured as reduction oftumor mass or inhibition of tumor mass increase. In addition to tumorresponse, an increase of overall survival, progress-free survival, ortime to tumor recurrence or a reduction of adverse effect also can beused clinically as a desired treatment effect.

(B) Treatments

The present disclosure is reflected in and substantiated by experimentalevidence that, in keeping with the inventors' discovery, bacteriallyderived and intact minicells or intact killed bacterial cells, whenadministered to a tumor patient along with an agent that increases thelevel of IFN-gamma, can achieve a therapeutic efficacy that issurprisingly greater than when the minicells or killed bacterial cellsare administered alone.

(C) Anti-Neoplastic Agents

The phrase “anti-neoplastic agent” denotes a drug, whether chemical orbiological, that prevents or inhibits the growth, development,maturation, or spread of neoplastic cells.

In the context of this disclosure, selecting an anti-neoplastic agentfor treating a given tumor patient depends on several factors, inkeeping with conventional medical practice. These factors include butare not limited to the patient's age, the stage of the tumor, andwhatever previous therapy the patient may have received.

In accordance with the disclosure, a drug can be selected from one ofthe classes detailed below, for packaging into intact, bacteriallyderived minicells, which then are administered to treat a tumor. Thesedrugs can also be synthetic analogs designed from drug design anddiscovery efforts.

-   -   Polyfunctional alkylating agents, exemplified by        Cyclophosphamide (Cytoxan), Mechlorethamine, Melphalan        (Alkeran), Chlorambucil (Leukeran), Thiopeta (Thioplex),        Busulfan (Myleran).    -   Alkylating drugs, exemplified by Procarbazine (Matulane),        Dacarbazine (DTIC), Altretamine (Hexalen), Clorambucil,        Cisplatin (Platinol), Carboplatin, Ifosafamide, Oxaliplatin.    -   Antimetabolites, exemplified by Methotrexate (MTX),        6-Thiopurines (Mercaptopurine [6-MP], Thioguanine [6-TG]),        Mercaptopurine (Purinethol), Thioguanine, Fludarabine phosphate,        Cladribine: (Leustatin), Pentostatin, Flurouracil (5-FU),        Cytarabine (ara-C), Azacitidine.    -   Plant alkaloids, terpenoids and topoisomerase inhibitors,        exemplified by Vinblastine (Velban), Vincristine (Oncovin),        Vindesine, Vinorelbine, Podophyllotoxins (etoposide {VP-16} and        teniposide {VM-26}), Camptothecins (topotecan and irinotecan),        Taxanes such as Paclitaxel (Taxol) and Docetaxel (Taxotere).    -   Antibiotics, exemplified by Doxorubicin (Adriamycin, Rubex,        Doxil), Daunorubicin, Duocarmycin, Idarubicin, Dactinomycin        (Cosmegen), Plicamycin (Mithramycin), Mitomycin: (Mutamycin),        Bleomycin (Blenoxane).    -   Hormonal agents, exemplified by Estrogen and Androgen Inhibitors        (Tamoxifen and Flutamide), Gonadotropin-Releasing Hormone        Agonists (Leuprolide and Goserelin (Zoladex)), Aromatase        Inhibitors (Aminoglutethimide and Anastrozole (Arimidex)).    -   Miscellaneous Anticancer Drugs, exemplified by Amsacrine,        Asparaginase (El-spar), Hydroxyurea, Mitoxantrone (Novantrone),        Mitotane (Lysodren), Maytansinoid, Retinoic acid Derivatives,        Bone Marrow Growth Factors (sargramostim and filgrastim),        Amifostine.    -   Agents disrupting folate metabolism, e.g., Pemetrexed.    -   DNA hypomethylating agents, e.g., Azacitidine, Decitabine.    -   Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)        pathway inhibitors, such as Iniparib, Olaparib, Veliparib.    -   PI3K/Akt/mTOR pathway inhibitors, e.g., Everolimus.    -   Histone deacetylase (HDAC) inhibitors, e.g., Vorinostat,        Entinostat (SNDX-275), Mocetinostat (MGCD0103), Panobinostat        (LBH589), Romidepsin, Valproic acid.    -   Cyclin-dependent kinase (CDK) inhibitors, e.g., Flavopiridol,        Olomoucine, Roscovitine, Kenpaullone, AG-024322 (Pfizer),        Fascaplysin, Ryuvidine, Purvalanol A, NU2058, BML-259, SU 9516,        PD-0332991, P276-00.    -   Heat shock protein (HSP90) inhibitors, e.g., Geldanamycin,        Tanespimycin, Alvespimycin, Radicicol, Deguelin, BIIB021.    -   Murine double minute 2 (MDM2) inhibitors, e.g., Cis-imidazoline,        Benzodiazepinedione, Spiro-oxindoles, Isoquinolinone, Thiophene,        5-Deazaflavin, Tryptamine.    -   Anaplastic lymphoma kinase (ALK) inhibitors, e.g.,        Aminopyridine, Diaminopyrimidine, Pyridoisoquinoline,        Pyrrolopyrazole, Indolocarbazole, Pyrrolopyrimidine,        Dianilinopyrimidine.    -   Poly [ADPribose] polymerase (PARP) inhibitors, illustrated by        Benzamide, Phthalazinone, Tricyclic indole, Benzimidazole,        Indazole, Pyrrolocarbazole, Phthalazinone, Isoindolinone.

Active agents useable in the present disclosure are not limited to thosedrug classes or particular agents enumerated above. Different discoveryplatforms continue to yield new agents that are directed at uniquemolecular signatures of cancer cells; indeed, thousands of such chemicaland biological drugs have been discovered, only some of which are listedhere. Yet, the surprising capability of intact, bacterially derivedminicells and killed bacterial cells to accommodate packaging of adiverse variety of active agents, hydrophilic or hydrophobic, means thatessentially any such drug, when packaged in minicells, has the potentialto treat a cancer, pursuant to the findings in the present disclosure.

Likewise illustrative of the class of anti-neoplastic agents areradionuclides, chemotherapy drugs, and functional nucleic acids,including but not limited to regulatory RNAs.

1. Radionuclides

A “radionuclide” is an atom with an unstable nucleus, i.e., onecharacterized by excess energy available to be imparted either to anewly created radiation particle within the nucleus or to an atomicelectron. Therefore, a radionuclide undergoes radioactive decay, andemits gamma ray(s) and/or subatomic particles. Numerous radionuclidesare known in the art, and a number of them are known to be suitable formedical use, such as yttrium-90, technetium-99m, iodine-123, iodine-124,iodine-125, iodine-131, rubidium-82, thallium-201, gallium-67,fluorine-18, xenon-133, and indium-111.

Radionuclides have found extensive use in nuclear medicine, particularlyas beta-ray emitters for damaging tumor cells. Radionuclides aresuitably employed, therefore, as anti-neoplastic agents in the presentdisclosure.

Radionuclides can be associated with intact, bacterially derivedminicells by any known technique. Thus, a protein or otherminicell-surface moiety (see below) can be labeled with a radionuclide,using a commercially available labeling means, such as use of PierceIodination reagent, a product of Pierce Biotechnology Inc. (Rockford,Ill.), detailed in Rice et al., Semin. Nucl. Med. 41, 265-282 (2011).Alternatively, radionuclides can be incorporated into proteins that areinside minicells.

In the latter situation, a minicell-producing bacterial strain istransformed with plasmid DNA encoding foreign protein. When minicellsare formed during asymmetric cell division, several copies of theplasmid DNA segregates into the minicell cytoplasm. The resultant,recombinant minicells are incubated, in the presence of radiolabeledamino acids, under conditions such that foreign protein expressed insidethe minicell, from the plasmid DNA, incorporates theradionuclide-carrying amino acids. Pursuant to the protocol ofClark-Curtiss and Curtiss, Methods Enzymol. 101: 347-362 (1983), forinstance, recombinant minicells are incubated in minimal growth mediumthat contains ³⁵S-methionine, whereby newly expressed, plasmid-encodedproteins incorporate the ³⁵S-methionine. A similar approach can be usedin order that recombinant minicells become packaged with otherradiolabels, as desired.

Oligosaccharides on the minicell surface also can be radiolabeled using,for example, well-established protocols described by Fukuda, Curr.Protocols Molec. Biol. (Suppl. 26), 17.5.1-17.5.8 (1994). Illustrativeof such oligosaccharides that are endemic to minicells is theO-polysaccharide component of the lipopolysaccharide (LPS) found on thesurface of minicells derived from Gram-negative bacteria (see below).

A preferred methodology in this regard is to radiolabel a bispecificantibody that is used to target minicells to specific tumors. Seesection G, infra, and patent publication US 2007/0237744, the contentsof which are incorporated herein by reference. That is, the bispecificantibody “coated” on a minicell exposes a significant amount ofadditional surface protein for radiolabeling. Accordingly, it ispossible to achieve a higher specific activity of the radiolabelassociated with the antibody-coated minicell. By contrast, theradiolabeling of non-coated minicells, i.e., when the radionuclidelabels only endemic moieties, can result in weaker labeling (lowerspecific activity). In one embodiment, this weaker labeling is thoughtto occur because the outer membrane-associated proteins of minicellsderived from Gram-negative bacteria are masked by LPS, which, as furtherdiscussed below, comprises long chains of O-polysaccharide covering theminicell surface.

For treating a tumor, a composition of the disclosure would be deliveredin a dose or in multiple doses that in toto affords a level of in-tumorirradiation that is sufficient at least to reduce tumor mass, if noteliminate the tumor altogether. The progress of treatment can bemonitored along this line, on a case-by-case basis. In general terms,however, the amount of radioactivity packaged in the compositiontypically will be on the order of about 30 to 50 Gy, although theinvention also contemplates a higher amount of radioactivity, say, about50 to 200 Gy, which gives an overall range between about 30 Gy and about200 Gy.

In some instances the amount of radioactivity packaged in thecomposition can be even lower than mentioned above, given the highlyefficient and specific delivery of the minicell-bourne radionuclides toa tumor. Accordingly, in one aspect the composition contains from about20 to 40 Gy, or about 10 to 30 Gy, or about 1 to about 20 Gy, or lessthan 10 Gy.

2. Chemotherapy Drugs

An anti-neoplastic agent employed in the present disclosure also can bea chemotherapy drug. In this description, “chemotherapeutic drug,”“chemotherapeutic agent,” and “chemotherapy” are employedinterchangeably to connote a drug that has the ability to kill ordisrupt a neoplastic cell. A chemotherapeutic agent can be a smallmolecule drug or a biologic drug, as further detailed below.

The “small molecule drug” subcategory encompasses compoundscharacterized by having (i) an effect on a biological process and (ii) alow molecular weight as compared to a protein or polymericmacromolecule. Small molecule drugs typically are about 800 Daltons orless, with a lower limit of about 150 Daltons, as illustrated byTemodar® (temozolomide), at about 194 Daltons, which is used to treatgliaoblastoma multiforme and other types of brain cancer. In thiscontext “about” indicates that the qualified molecular-weight value issubject to variances in measurement precision and to experimental erroron the order of several Daltons or tens of Daltons. Thus, a smallmolecule drug can have a molecular weight of about 900 Daltons or less,about 800 or less, about 700 or less, about 600 or less, about 500 orless, or about 400 Daltons or less, e.g., in the range of about 150 toabout 400 Daltons. More specifically, a small molecule drug can have amolecular weight of about 400 Daltons or more, about 450 Daltons ormore, about 500 Daltons or more, about 550 Daltons or more, about 600Daltons or more, about 650 Daltons or more, about 700 Daltons or more,or about 750 Daltons or more. In another embodiment, the small moleculedrug packaged into the minicells has a molecular weight between about400 and about 900 Daltons, between about 450 and about 900 Daltons,between about 450 and about 850 Daltons, between about 450 and about 800Daltons, between about 500 and about 800 Daltons, or between about 550and about 750 Daltons.

Specifically, suitable small molecule drugs include but are not limitedto nitrogen mustards, nitrosorueas, ethyleneimine, alkane sulfonates,tetrazine, platinum compounds, pyrimidine analogs, purine analogs,anti-metabolites, folate analogs, anthracyclines, taxanes, vincaalkaloids, and topoisomerase inhibitors, inter alia. Accordingly, asmall molecule drug for use in the present invention can be selectedfrom among any of the following, inter alia: enediynes, such asdynemicin A, unicalamycin, calicheamicin γ1 and calicheamicin θ1;meayamicin, a synthetic analog of FR901464; benzosuberene derivatives asdescribed, for example, by Tanpure et al., Bioorg. Med. Chem. 21:8019-32 (2013); auristatins, such as auristatin E, mono-methylauristatin E (MMAE), and auristatin F, which are synthetic analogs ofdolastatin; duocarmysins such as duocarmycin SA and CC-1065; maytansineand its derivatives (maytansinoids), such as DM1 and DM4; irinotecan(Camptosar®) and other topoisomerase inhibitors, such as topotecan,etoposide, mitoxantrone and teniposide; and yatakemycin, the synthesisof which is detailed by Okano et al., J. Am. Chem. Soc. 128: 7136-37(2006).

More particularly, any one or more or all of the specific small moleculedrugs detailed in this paragraph are illustrative of those suitable foruse in this invention: actinomycin-D, alkeran, ara-C, anastrozole,BiCNU, bicalutamide, bisantrene, bleomycin, busulfan, capecitabine(Xeloda®), carboplatin, carboplatinum, carmustine, CCNU, chlorambucil,cisplatin, cladribine, CPT-11, cyclophosphamide, cytarabine, cytosinearabinoside, cytoxan, dacarbazine, dactinomycin, daunorubicin,dexrazoxane, docetaxel, doxorubicin, DTIC, epirubicin, ethyleneimine,etoposide, floxuridine, fludarabine, fluorouracil, flutamide,fotemustine, gemcitabine, hexamethylamine, hydroxyurea, idarubicin,ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan,mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone,oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin,procarbazine, streptozocin, STI-571, tamoxifen, temozolomide,teniposide, tetrazine, thioguanine, thiotepa, tomudex, topotecan,treosulphan, trimetrexate, vinblastine, vincristine, vindesine,vinorelbine, and VP-16.

For purposes of this description a “biologic drug” is defined, bycontrast, to denote any biologically active macromolecule that can becreated by a biological process, exclusive of “functional nucleicacids,” discussed below, and polypeptides that by size qualify as smallmolecule drugs, as defined above. The “biologic drug” subcategory thusis exclusive of and does not overlap with the small molecule drug andfunctional nucleic acid subcategories. Illustrative of biologic drugsare therapeutic proteins and antibodies, whether natural or recombinantor synthetically made, e.g., using the tools of medicinal chemistry anddrug design.

Certain molecules that are designed for chemotherapeutic purposesnevertheless fail during pre-clinical or clinical trials due tounacceptable toxicity or other safety concerns. The present inventorshave shown that packaging a chemotherapy drug in a minicell, followed bysystemic delivery to a tumor patient, results in delivery of the drug totumor cells. Further, even after the tumor cells are broken up and thedrug-containing cytoplasm is released to the nearby normal tissue, theresult is not toxicity to normal tissue. This is because the drugalready is bound to the tumor cellular structures, such as DNA, and canno longer attack normal cells. Accordingly, the present invention isparticularly useful for delivery of highly toxic chemotherapy drugs to atumor patient.

The phrases “highly toxic chemotherapy drug” and “supertoxicchemotherapy drug” in this description refer to chemotherapy drugs thathave a relatively low lethal dose to normal cells as compared to theireffective dose for cancer cells. Thus, in one aspect a highly toxicchemotherapy drug has a median lethal dose (LD₅₀) that is lower than itsmedian effective dose (ED₅₀) for a targeted cancer such as (1) a cancertype for which the drug is designed, (2) the first cancer type in whicha pre-clinical or clinical trial is run for that drug, or (3) the cancertype in which the drug shows the highest efficacy among all testedcancers. For instance, a highly toxic chemotherapy drug can have an LD₅₀that is lower than about 500%, 400%, 300%, 250%, 200%, 150%, 120%, or100% of the ED₅₀ of the drug for a targeted cancer. In another aspect, ahighly toxic chemotherapy drug has a maximum sub-lethal dose (i.e., thehighest dose that does not cause serious or irreversible toxicity) thatis lower than its minimum effective dose for a targeted cancer, e.g.,about 500%, 400%, 300%, 250%, 200%, 150%, 120%, 100%, 90%, 80%, 70%, 60%or 50% of the minimum effective dose.

According to one embodiment of the present description, therefore, atumor in a subject is treated by a method comprising administeringsystemically a therapeutically effective amount of a compositioncomprised of a plurality of intact, bacterially derived minicells, eachof which encompasses a highly toxic chemotherapy drug. Maytansinoids andduocarmycins, discussed below, are representative of the class ofsupertoxic chemotherapy drugs thus employed.

Suitable cancer chemotherapy drugs in the context include nitrogenmustards, nitrosorueas, ethyleneimine, alkane sulfonates, tetrazine,platinum compounds, pyrimidine analogs, purine analogs, antimetabolites,folate analogs, anthracyclines, taxanes, vinca alkaloids, topoisomeraseinhibitors, and hormonal agents, inter alia.

Chemotherapy drugs that are illustrative of the small molecule drugsubcategory are actinomycin-D, alkeran, ara-C, anastrozole, BiCNU,bicalutamide, bleomycin, busulfan, capecitabine (Xeloda®), carboplatin,carboplatinum, carmustine, CCNU, chlorambucil, cisplatin, cladribine,CPT-11, cyclophosphamide, cytarabine, cytosine arabinoside, cytoxan,dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel,doxorubicin, DTIC, epirubicin, ethyleneimine, etoposide, floxuridine,fludarabine, fluorouracil, flutamide, fotemustine, gemcitabine,hexamethylamine, hydroxyurea, idarubicin, ifosfamide, irinotecan,lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate,mitomycin, mitotane, mitoxantrone, oxaliplatin, paclitaxel, pamidronate,pentostatin, plicamycin, procarbazine, streptozocin, STI-571, tamoxifen,temozolomide, teniposide, tetrazine, thioguanine, thiotepa, tomudex,topotecan, treosulphan, trimetrexate, vinblastine, vincristine,vindesine, vinorelbine, and VP-16.

Maytansinoids (molecular weight: ˜738 Daltons) are a group of chemicalderivatives of maytansine, having potent cytotoxicity. Althoughconsidered unsafe for human patient use, due to toxicity concerns,maytansinoids are suitable for delivery to tumor patients via minicells,pursuant to the present invention.

Duocarmycins (molecular weight: ˜588 Daltons) are a series of relatednatural products, first isolated from Streptomyces bacteria. They alsohave potent cytotoxicity but are considered as unsafe for human use.Like maytansinoids, duocarmycins are suitable chemotherapy drugs for usein the invention.

Likewise illustrative are compounds in the class of morpholinylanthracycline derivatives described in international patent applicationWO1998/002446. Among such derivatives are nemorubicin(3′-deamino-3′-[2(S)-methoxy-4-morpholinyl]doxorubicin), a/k/a MMDX, andits major metabolite PNU-159682(3′-deamino-3″-4′-anhydro-[2″(S)-methoxy-3″(R)-hydroxy-4″-morpholinyl]doxorubicin), the structural formula of which is shown below, as well asthese four other such derivatives described in U.S. Pat. No. 8,470,984,the contents of which are incorporated here by reference:3′-deamino-3″-4′-anhydro-[2″(S)-methoxy-3″(R)-hydroxy-4″-morpholinyl]idarubicin;3′-deamino-3″-4′-anhydro-[2″(S)-methoxy-3″(R)-hydroxy-4″-morpholinyl]daunorubicin;3′-deamino-3″-4′-anhydro-[2″(S)-methoxy-3″(R)-hydroxy-4″-morpholinyl]-caminomycin;and3′-deamino-3″-4′-anhydro-[2″(S)-ethoxy-3″(R)-hydroxy-4″-morpholinyl]d-oxorubicin.

A pharmaceutically acceptable acid addition salt of any of theaforementioned derivatives also is a member, pursuant to the invention,of this group of autofluorescent morpholinyl anthracycline derivatives.

The subcategory of biologic chemotherapy drugs includes, withoutlimitation, asparaginase, AIN-457, bapineuzumab, belimumab, brentuximab,briakinumab, canakinumab, cetuximab, dalotuzumab, denosumab,epratuzumab, estafenatox, farletuzumab, figitumumab, galiximab,gemtuzumab, girentuximab (WX-G250), ibritumomab, inotuzumab, ipilimumab,mepolizumab, muromonab-CD3, naptumomab, necitumumab, nimotuzumab,ocrelizumab, ofatumumab, otelixizumab, ozogamicin, pagibaximab,panitumumab, pertuzumab, ramucirumab, reslizumab, rituximab, REGN88,solanezumab, tanezumab, teplizumab, tiuxetan, tositumomab, trastuzumab(Herceptin®), tremelimumab, vedolizumab, zalutumumab, and zanolimumab.

The composition can contain at most about 1 mg of the chemotherapeuticdrug. Alternatively, the amount of the chemotherapeutic drug can be atmost about 750 μs, 500 μs, 250 μg, 100 μs, 50 μs, 10 μs, 5 μg, 1 μg, 0.5μg, or 0.1 In another aspect, the composition contains achemotherapeutic drug having an amount of less than about 1/1,000, oralternatively less than about 1/2,000, 1/5,000, 1/10,000, 1/20,000,1/50,000, 1/100,000, 1/200,000 or 1/500,000 of the therapeuticallyeffective amount of the drug when used without being packaged to intominicells. Pursuant to yet another aspect of the disclosure, thecomposition can contain at least about 1 nmol of the chemotherapeuticdrug. Accordingly, the disclosure also encompasses embodiments where theamount of the chemotherapeutic drug is at least about 2 nmol, about 3nmol, about 4 nmol, about 5 nmol, about 10 nmol, about 20 nmol, about 50nmol, about 100 nmol, and about 800 nmol, respectively.

3. Functional Nucleic Acids

“Functional nucleic acid” refers to a nucleic acid molecule that, uponintroduction into a host cell, specifically interferes with expressionof a protein. With respect to treating a tumor, in accordance with thedisclosure, it is preferable that a functional nucleic acid payloaddelivered to tumor cells via intact, bacterially derived minicellsinhibits a gene that promotes tumor cell proliferation, angiogenesis orresistance to chemotherapy and/or that inhibits apoptosis or cell-cyclearrest; i.e., a “tumor-promoting gene.”

It is generally the case that functional nucleic acid molecules used inthis disclosure have the capacity to reduce expression of a protein byinteracting with a transcript for a protein. This category of minicellpayload for the disclosure includes regulatory RNAs, such as siRNA,shRNA, short RNAs (typically less than 400 bases in length), micro-RNAs(miRNAs), ribozymes and decoy RNA, antisense nucleic acids, and LincRNA,inter alia. In this regard, “ribozyme” refers to an RNA molecule havingan enzymatic activity that can repeatedly cleave other RNA molecules ina nucleotide base sequence-specific manner. “Antisense oligonucleotide”denotes a nucleic acid molecule that is complementary to a portion of aparticular gene transcript, such that the molecule can hybridize to thetranscript and block its translation. An antisense oligonucleotide cancomprise RNA or DNA. The “LincRNA” or “long intergenic non-coding RNA”rubric encompasses non-protein coding transcripts longer than 200nucleotides. LincRNAs can regulate the transcription, splicing, and/ortranslation of genes, as discussed by Khalil et al., Proc Nat'l Acad.USA 106: 11667-72 (2009), for instance.

Each of the types of regulatory RNA can be the source of functionalnucleic acid molecule that inhibits a tumor-promoting gene as describedabove and, hence, that is suitable for use according to the presentdisclosure. Thus, in one preferred embodiment of the disclosure theintact minicells carry siRNA molecules mediating a post-transcriptional,gene-silencing RNA interference (RNAi) mechanism, which can be exploitedto target tumor-promoting genes. For example, see MacDiarmid et al.,Nature Biotech. 27: 645-51 (2009) (antibody-presenting minicellsdeliver, with chemotherapy drug, siRNAs that counter developingresistance to drug), and Oh and Park, Advanced Drug Delivery Rev. 61:850-62 (2009) (delivery of therapeutic siRNAs to treat breast, ovarian,cervical, liver, lung and prostate cancer, respectively).

As noted, “siRNA” generally refers to double-stranded RNA molecules fromabout 10 to about 30 nucleotides long that are named for their abilityspecifically to interfere with protein expression. Preferably, siRNAmolecules are 12-28 nucleotides long, more preferably 15-25 nucleotideslong, still more preferably 19-23 nucleotides long and most preferably21-23 nucleotides long. Therefore, siRNA molecules can be 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28 or 29 nucleotidesin length.

The length of one strand designates the length of an siRNA molecule. Forinstance, an siRNA that is described as 21 ribonucleotides long (a21-mer) could comprise two opposing strands of RNA that anneal for 19contiguous base pairings. The two remaining ribonucleotides on eachstrand would form an “overhang.” When an siRNA contains two strands ofdifferent lengths, the longer of the strands designates the length ofthe siRNA. For instance, a dsRNA containing one strand that is 21nucleotides long and a second strand that is 20 nucleotides long,constitutes a 21-mer.

Tools to assist the design of siRNA specifically and regulatory RNAgenerally are readily available. For instance, a computer-based siRNAdesign tool is available on the internet at www.dharmacon.com.

In another preferred embodiment, the intact minicells of the presentdisclosure carry miRNAs, which, like siRNA, are capable of mediating apost-transcriptional, gene-silencing RNA interference (RNAi) mechanism.Also like siRNA, the gene-silencing effect mediated by miRNA can beexploited to target tumor-promoting genes. For example, see Kota et al.,Cell 137: 1005-17 (2009) (delivery of a miRNA via transfection resultedin inhibition of cancer cell proliferation, tumor-specific apoptosis anddramatic protection from disease progression without toxicity in murineliver cancer model), and Takeshita, et al., Molec. Ther. 18: 181-87(2010) (delivery of synthetic miRNA via transient transfection inhibitedgrowth of metastatic prostate tumor cells on bone tissues).

Although both mediate RNA interference, miRNA and siRNA have noteddifferences. In this regard, “miRNA” generally refers to a class of 17-to 27-nucleotide single-stranded RNA molecules (instead ofdouble-stranded as in the case of siRNA). Therefore, miRNA molecules canbe 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 nucleotides in length.Preferably, miRNA molecules are 21-25 nucleotide long.

Another difference between miRNAs and siRNAs is that the formergenerally do not fully complement the mRNA target. On the other hand,siRNA must be completely complementary to the mRNA target. Consequently,siRNA generally results in silencing of a single, specific target, whilemiRNA is promiscuous.

Additionally, although both are assembled into RISC (RNA-inducedsilencing complex), siRNA and miRNA differ in their respective initialprocessing before RISC assembly. These differences are described indetail in Chu et al., PLoS Biology 4: 1122-36 (2006), and Gregory etal., Methods in Molecular Biology 342: 33-47 (2006).

A number of databases serve as miRNA depositories. For example, seemiRBase (www.mirbase.org) and tarbase(http://diana.cslab.ece.ntua.gr/DianaToolsNew/index.php?r=tarbase/index).In conventional usage, miRNAs typically are named with the prefix“-mir,” combined with a sequential number. For instance, a new miRNAdiscovered after mouse mir-352 will be named mouse “mir-353.”

Again, tools to assist the design of regulatory RNA including miRNA arereadily available. In this regard, a computer-based miRNA design tool isavailable on the internet at wmd2.weigelworld.org/cgi-bin/mirnatools.pl.

As noted above, a functional nucleic acid employed in the disclosure caninhibit a gene that promotes tumor cell proliferation, angiogenesis orresistance to chemotherapy. The inhibited gene also can itself inhibitapoptosis or cell cycle arrest. Examples of genes that can be targetedby a functional nucleic acid are provided below.

Functional nucleic acids of the disclosure preferably target the gene ortranscript of a protein that promotes drug resistance, inhibitsapoptosis or promotes a neoplastic phenotype. Successful application offunctional nucleic acid strategies in these contexts have been achievedin the art, but without the benefits of minicell vectors. See, e.g.,Sioud, Trends Pharmacol. Sci. 25: 22-8 (2004), Caplen, Expert Opin.Biol. Ther. 3: 575-86 (2003), Nieth et al., FEBS Lett. 545: 144-50(2003), Caplen and Mousses, Ann. NY Acad. Sci. 1002: 56-62 (2003),Duxbury et al., Ann. Surg. 240: 667-74 (2004), Yague et al., Gene Ther.11: 1170-74 (2004), and Duan et al., Mol. Cancer Ther. 3: 833-8 (2004).

Proteins that contribute to drug resistance constitute preferred targetsof functional nucleic acids. The proteins may contribute to acquireddrug resistance or intrinsic drug resistance. When diseased cells, suchas tumor cells, initially respond to drugs, but become refractory onsubsequent treatment cycles, the resistant phenotype is acquired. Usefultargets involved in acquired drug resistance include ATP bindingcassette transporters such as P-glycoprotein (P-gp, P-170, PGY1, MDR1,ABCB1, MDR-associated protein, Multidrug resistance protein 1), MDR-2and MDR-3. MRP2 (multi-drug resistance associated protein), BCR-ABL(breakpoint cluster region—Abelson protooncogene), a STI-571resistance-associated protein, lung resistance-related protein,cyclooxygenase-2, nuclear factor kappa, XRCC1 (X-ray cross-complementinggroup 1), ERCC1 (excision cross-complementing gene), GSTP1 (glutathioneS-transferase), mutant β-tubulin, and growth factors such as IL-6 areadditional targets involved in acquired drug resistance.

Particularly useful targets that contribute to drug resistance includeATP binding cassette transporters such as P-glycoprotein, MDR-2, MDR-3,BCRP, APT11a, and LRP.

Useful targets also include proteins that promote apoptosis resistance.These include Bcl-2 (B cell leukemia/lymphoma), Bcl-X_(L), A1/Bfl 1,focal adhesion kinase, dihydrodiol dehydrogenase, and p53 mutantprotein.

Useful targets further include oncogenic and mutant tumor suppressorproteins. Illustrative of these are β-Catenin, PKC-α (protein kinase C),C-RAF, K-Ras (V12), DP97 Dead box RNA helicase, DNMT1 (DNAmethyltransferase 1), FLIP (Flice-like inhibitory protein), C-Sfc,53BPI, Polycomb group protein EZH2 (Enhancer of zeste homologue), ErbB1,HPV-16 E5 and E7 (human papillomavirus early 5 and early 7), Fortilin &MCI1P (Myeloid cell leukemia 1 protein), DIP13α (DDC interacting protein13a), MBD2 (Methyl CpG binding domain), p21, KLF4 (Kruppel-like factor4), tpt/TCTP (Translational controlled tumor protein), SPK1 and SPK2(Sphingosine kinase), P300, PLK1 (Polo-like kinase-1), Trp53, Ras,ErbB1, VEGF (Vascular endothelial growth factor), BAG-1 (BCL2-associatedathanogene 1), MRP2, BCR-ABL, STI-571 resistance-associated protein,lung resistance-related protein, cyclooxygenase-2, nuclear factor kappa,XRCC1, ERCC1, GSTP1, mutant β-tubulin, and growth factors.

Also useful as targets are global regulatory elements exemplified by thecytoplasmic polyadenylation element binding proteins (CEPBs). Forinstance, CEPB4 is overexpressed in glioblastoma and pancreatic cancers,where the protein activates hundreds of genes associated with tumorgrowth, and it is not detected in healthy cells (Oritz-Zapater et al.,Nature Medicine, doi: 10.1038/nm.2540 (published on-line Dec. 4, 2011)).In accordance with the present description, therefore, treatment of aglioblastoma could be effected via administration of a compositioncontaining intact, bacterially derived minicells that encompass an agentthat counters overexpression of CEPB4, such as an siRNA or otherfunctional nucleic acid molecule that disrupts CEPB4 expression by thetumor cells.

Further useful functional nucleic acids are those that are involved inDNA replication and repair. Examples include ribonucleotide reductase(RR), which is a potential therapeutic target for cancer because itcatalyzes the conversion of ribonucleoside 5′-diphosphates into theircorresponding 2′-deoxyribonucleoside 5′-triphosphates that are necessaryfor DNA replication and repair. See D'Angiolella et al., Cell:149:1023-34 (2012). Human RR comprises two subunits, RRM1 and RRM2, andfunctional nucleic acids that target both subunits are useful in thepresent invention. A further example of useful functional nucleic acidsinclude replication protein A (RPA), a trimeric complex composed of70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits, which isessential for DNA replication in all organisms. See Iftode et al., Crit.Rev. Biochem. Mol. Biol. 34: 141-80 (1999).

(D) Tumors

The compositions and methods of the present disclosure are useful intreating a variety of tumor types, not limited to a particular kind.This is because the minicells or killed bacterial cells can packagedifferent anti-neoplastic agents and, in particular when attached with abispecific ligand specific to different tumor cells, can target cells ofdifferent tumor types. In addition, the ability of minicells or killedbacterial cells, in combination with IFN-gamma, are expected to be ableto stimulate immune response to any tumor cells.

In accordance with one embodiment of the disclosure, the presentcompositions and methods are used in treating one or more cancersselected from adrenal cancer, anal cancer, aplastic anemia, bile ductcancer, bladder cancer, bone cancer, brain/CNS tumors in adults,brain/CNS tumors in children, breast cancer, breast cancer in men,cancer in children, cancer of unknown primary, Castleman disease,cervical cancer, colon/rectum cancer, endometrial cancer, esophaguscancer, Ewing family of tumors, eye cancer, gallbladder cancer,gastrointestinal carcinoid tumors, gastrointestinal stromal tumor(gist), gestational trophoblastic disease, Hodgkin disease, Kaposisarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia,leukemia—acute lymphocytic (ALL) in adults, leukemia—acute myeloid(AML), leukemia—chronic lymphocytic (CLL), leukemia—chronic myeloid(cml), leukemia—chronic myelomonocytic (CMML), leukemia in children,liver cancer, lung cancer, lung cancer—non-small cell, lung cancer—smallcell, lung carcinoid tumor, lymphoma, lymphoma of the skin, malignantmesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavityand paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma,non-Hodgkin lymphoma, non-Hodgkin lymphoma in children, oral cavity andoropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,penile cancer, pituitary tumors, prostate cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcoma—adult soft tissuecancer, skin cancer, skin cancer—basal and squamous cell, skincancer—melanoma, small intestine cancer, stomach cancer, testicularcancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer,vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

In one embodiment, the compositions and methods are suitable fortreating brain tumor. There are more than 120 types of brain tumors.Most medical institutions use the World Health Organization (WHO)classification system to identify brain tumors. The WHO classifies braintumors by cell origin and how the cells behave, from the leastaggressive (benign) to the most aggressive (malignant). Some tumor typesare assigned a grade, ranging from Grade I (least malignant) to Grade IV(most malignant), which signifies the rate of growth. There arevariations in grading systems, depending on the tumor type. Theclassification and grade of an individual tumor help predict its likelybehavior. The most frequently diagnosed types include acoustic neuroma,astrocytoma (including Grade I—pilocytic astrocytoma, Grade II—low-gradeastrocytoma, Grade III—anaplastic astrocytoma, and Grade IV—glioblastoma(GBM)), chordoma, CNS lymphoma, craniopharyngioma, other gliomas (brainstem glioma, ependymoma, mixed glioma, optic nerve glioma andsubependymoma), medulloblastoma, meningioma, metastatic brain tumors,oligodendroglioma, pituitary tumors, primitive neuroectodermal (PNET),other brain-related conditions, and schwannoma.

Among children, these brain tumor types are more common: brain stemglioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma(JPA), medulloblastoma, optic nerve glioma, pineal tumor, primitiveneuroectodermal tumors (PNET), and rhabdoid tumor.

(E) Minicells and Killed Bacterial Cells

“Minicell” refers to a derivative of a bacterial cell that is lacking inchromosomes (“chromosome-free”) and is engendered by a disturbance inthe coordination, during binary fission, of cell division with DNAsegregation. Minicells are distinct from other small vesicles, such asso-called “membrane blebs” (˜0.2 μm or less in size), which aregenerated and released spontaneously in certain situations but which arenot due to specific genetic rearrangements or episomal gene expression.By the same token, intact minicells are distinct from bacterial ghosts,which are not generated due to specific genetic rearrangements orepisomal gene expression. Bacterially derived minicells employed in thisdisclosure are fully intact and thus are distinguished from otherchromosome-free forms of bacterial cellular derivatives characterized byan outer or defining membrane that is disrupted or degraded, evenremoved. See U.S. Pat. No. 7,183,105 at column 111, lines 54 et seq. Theintact membrane that characterizes the minicells of the presentdisclosure allows retention of the therapeutic payload within theminicell until the payload is released, post-uptake, within a tumorcell.

The minicell employed in this disclosure can be prepared from bacterialcells, such as E. coli and S. typhymurium. Prokaryotic chromosomalreplication is linked to normal binary fission, which involves mid-cellseptum formation. In E. coli, for example, mutation of min genes, suchas minCD, can remove the inhibition of septum formation at the cellpoles during cell division, resulting in production of a normal daughtercell and an chromosome-less minicell. See de Boer et al., J. Bacteriol.174(1): 63-70 (1992); Raskin & de Boer, J. Bacteriol. 181: 6419-6424(1999); Hu & Lutkenhaus, Mol. Microbio. 34(1): 82-90 (1999); Harry, Mol.Microbiol. 40(4): 795-803 (2001).

In addition to min operon mutations, chromosome-less minicells also aregenerated following a range of other genetic rearrangements or mutationsthat affect septum formation, for example, in the divIVB1 in B.subtilis. See Reeve and Cornett, J. Virol. 15: 1308-16 (1975). Minicellsalso can be formed following a perturbation in the levels of geneexpression of proteins involved in cell division/chromosome segregation.For instance, over-expression of minE leads to polar division andproduction of minicells. Similarly, chromosome-less minicells can resultfrom defects in chromosome segregation, e.g., the smc mutation inBacillus subtilis (Britton et al., Genes Dev. 12: 1254-9 (1998)), thespoOJ deletion in B. subtilis (Ireton et al., J. Bacteriol. 176: 5320-29(1994)), the mukB mutation in E. coli (Hiraga et al., J. Bacteriol. 171:1496-1505 (1989)), and the parC mutation in E. coli (Stewart and D'Ari,J. Bacteriol. 174: 4513-6 (1992)). Further, CafA can enhance the rate ofcell division and/or inhibit chromosome partitioning after replication(Okada et al., J. Bacteriol. 176: 917-22 (1994)), resulting in formationof chained cells and chromosome-less minicells.

Accordingly, minicells can be prepared for the present disclosure fromany bacterial cell, be it of Gram-positive or Gram-negative origin dueto the conserved nature of bacterial cell division in these bacteria.Furthermore, the minicells used in the disclosure should possess intactcell walls (i.e., are “intact minicells”), as noted above, and should bedistinguished over and separated from other small vesicles, such asmembrane blebs, which are not attributable to specific geneticrearrangements or episomal gene expression.

In a given embodiment, the parental (source) bacteria for the minicellscan be Gram positive, or they can be Gram negative, as mentioned. In oneaspect, therefore, the parental bacteria are one or more selected fromTerra-/Glidobacteria (BV1), Proteobacteria (BV2), BV4 includingSpirochaetes, Sphingobacteria, and Planctobacteria. Pursuant to anotheraspect, the bacteria are one or more selected from Firmicutes (BV3) suchas Bacilli, Clostridia or Tenericutes/Mollicutes, or Actinobacteria(BV5) such as Actinomycetales or Bifidobacteriales.

Pursuant to the invention, killed bacterial cells are non-livingprokaryotic cells of bacteria, cyanobateria, eubacteria andarchaebacteria, as defined in the 2nd edition of BERGEY'S MANUAL OFSYSTEMATIC BIOLOGY. Such cells are deemed to be “intact” if they possessan intact cell wall and/or cell membrane and contain genetic material(nucleic acid) that is endogenous to the bacterial species. Methods ofpreparing killed bacterial cells are described, for instance, in U.S.patent application publication No. 2008/0038296, the contents of whichare incorporated here by reference.

In yet a further aspect, the bacteria are one or more selected fromEobacteria (Chloroflexi, Deinococcus-Thermus), Cyanobacteria,Thermodesulfobacteria, thermophiles (Aquificae, Thermotogae), Alpha,Beta, Gamma (Enterobacteriaceae), Delta or Epsilon Proteobacteria,Spirochaetes, Fibrobacteres, Chlorobi/Bacteroidetes,Chlamydiae/Verrucomicrobia, Planctomycetes, Acidobacteria,Chrysiogenetes, Deferribacteres, Fusobacteria, Gemmatimonadetes,Nitrospirae, Synergistetes, Dictyoglomi, Lentisphaerae Bacillales,Bacillaceae, Listeriaceae, Staphylococcaceae, Lactobacillales,Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Streptococcaceae,Clostridiales, Halanaerobiales, Thermoanaerobacterales, Mycoplasmatales,Entomoplasmatales, Anaeroplasmatales, Acholeplasmatales,Haloplasmatales, Actinomycineae, Actinomycetaceae, Corynebacterineae,Nocardiaceae, Corynebacteriaceae, Frankineae, Frankiaceae,Micrococcineae, Brevibacteriaceae, and Bifidobacteriaceae.

For pharmaceutical use, a composition of the disclosure should compriseminicells or killed bacterial cells that are isolated as thoroughly aspossible from immunogenic components and other toxic contaminants.Methodology for purifying bacterially derived minicells to remove freeendotoxin and parent bacterial cells are described in WO 2004/113507,which is incorporated by reference here in its entirety. Briefly, thepurification process achieves removal of (a) smaller vesicles, such asmembrane blebs, which are generally smaller than 0.2 μm in size, (b)free endotoxins released from cell membranes, and (c) parental bacteria,whether live or dead, and their debris, which are sources of freeendotoxins, too. Such removal can be implemented with, inter alia, a 0.2μm filter to remove smaller vesicles and cell debris, a 0.45 μm filterto remove parental cells following induction of the parental cells toform filaments, antibiotics to kill live bacterial cells, and antibodiesagainst free endotoxins.

Underlying the purification procedure is a discovery by the presentinventors that, despite the difference of their bacterial sources, allintact minicells are approximately 400 nm in size, i.e., larger thanmembrane blebs and other smaller vesicles and yet smaller than parentalbacteria. Size determination for minicells can be accomplished by usingsolid-state, such as electron microscopy, or by liquid-based techniques,e.g., dynamic light scattering. The size value yielded by each suchtechnique can have an error range, and the values can differ somewhatbetween techniques. Thus, the size of minicells in a dried state can bemeasured via electron microscopy as approximately 400 nm±50 nm. On theother hand, dynamic light scattering can measure the same minicells tobe approximately 500 nm±50 nm in size. Also, drug-packaged,ligand-targeted minicells can be measured, again using dynamic lightscattering, to be approximately 400 nm to 600 nm±50 nm.

This scatter of size values is readily accommodated in practice, e.g.,for purposes of isolating minicells from immunogenic components andother toxic contaminants, as described above. That is, an intact,bacterially derived minicell is characterized by cytoplasm surrounded bya rigid membrane, which gives the minicell a rigid, spherical structure.This structure is evident in transmission-electron micrographs, in whichminicell diameter is measured, across the minicell, between the outerlimits of the rigid membrane. This measurement provides theabove-mentioned size value of 400 nm±50 nm.

Another structural element of a killed bacterial cells or a minicellderived from Gram-negative bacteria is the O-polysaccharide component oflipopolysaccharide (LPS), which is embedded in the outer membrane viathe lipid A anchor. The component is a chain of repeatcarbohydrate-residue units, with as many as 70 to 100 repeat units offour to five sugars per chain. Because these chains are not rigid, in aliquid environment, as in vivo, they can adopt a waving, flexiblestructure that gives the general appearance of seaweed in a coral seaenvironment; i.e., the chains move with the liquid while remaininganchored to the minicell membrane.

Influenced by the O-polysaccharide component, dynamic light scatteringcan provide a value for minicell size of about 500 nm to about 600 nm,as noted above. Nevertheless, minicells from Gram-negative andGram-positive bacteria alike readily pass through a 0.45 μm filter,which substantiates an effective minicell size of 400 nm±50 nm. Theabove-mentioned scatter in sizes is encompassed by the present inventionand, in particular, is denoted by the qualifier “approximately” in thephrase “approximately 400 nm in size” and the like.

In relation to toxic contaminants, a composition of the disclosure cancontain less than about 350 EU free endotoxin. Illustrative in thisregard are levels of free endotoxin of about 250 EU, about 200 EU, about150 EU, about 100 EU, about 90 EU, about 80 EU, about 70 EU, about 60EU, about 50 EU, about 40 EU, about 30 EU, about 20 EU, about 15 EU,about 10 EU, about 9 EU, about 8 EU, about 7 EU, about 6 EU, about 5 EU,about 4 EU, about 3 EU, about 2 EU, about 1 EU, about 0.9 EU, about 0.8EU, about 0.7 EU, about 0.6 EU, about 0.5 EU, about 0.4 EU, about 0.3EU, about 0.2 EU, about 0.1 EU, about 0.05 EU, and about 0.01 EU,respectively.

A composition of the disclosure also can contain at least about 10⁹minicells or killed bacterial cells, e.g., at least about 1×10⁹, atleast about 2×10⁹, or at least about 5×10⁹. In some embodiments, thecomposition contains no more than about 10¹¹ minicells or killedbacterial cells, e.g., no more than about 1×10¹¹ or no more than about9×10¹⁰, or no more than about 8×10¹⁰

(F) Packaging an Anti-Neoplastic Agent into Minicells or KilledBacterial Cells

Anti-neoplastic agents, such as proteins and functional nucleic acids,that can be encoded by a nucleic acid, can be introduced into minicellsby transforming into the parental bacterial cell a vector, such as aplasmid, that encodes the anti-neoplastic agent. When a minicell isformed from the parental bacterial cell, the minicell retains certaincopies of the plasmid and/or the expression product, the anti-neoplasticagent. More details of packaging an expression product into a minicellis provided in WO 03/033519, the contents of which are incorporated intothe present disclosure in its entirety by reference.

Data presented in WO 03/033519 demonstrated, for example, thatrecombinant minicells carrying mammalian gene expression plasmids can bedelivered to phagocytic cells and to non-phagocytic cells. Theapplication also described the genetic transformation ofminicell-producing parent bacterial strains with heterologous nucleicacids carried on episomally-replicating plasmid DNAs. Upon separation ofparent bacteria and minicells, some of the episomal DNA segregated intothe minicells. The resulting recombinant minicells were readily engulfedby mammalian phagocytic cells and became degraded within intracellularphagolysosomes. Moreover, some of the recombinant DNA escaped thephagolysosomal membrane and was transported to the mammalian cellnucleus, where the recombinant genes were expressed.

Nucleic acids also can be packaged into minicells directly. Thus, anucleic acid can be packaged directly into intact minicells byco-incubating a plurality of intact minicells with the nucleic acid in abuffer. The buffer composition can be varied, as a function ofconditions well known in this field, in order to optimize the loading ofthe nucleic acid in the intact minicells. The buffer also may be variedin dependence on the nucleotide sequence and the length of the nucleicacid to be loaded in the minicells. Exemplary buffer suitable forloading includes, but is not limited to, phosphate buffered saline(PBS). Once packaged, the nucleic acid remains inside the minicell andis protected from degradation. Prolonged incubation studies withsiRNA-packaged minicells incubated in sterile saline have shown, forexample, no leakage of siRNAs.

In other embodiments, multiple nucleic acids directed to different mRNAtargets can be packaged in the same minicell. Such an approach can beused to combat drug resistance and apoptosis resistance. For instance,cancer patients routinely exhibit resistance to chemotherapeutic drugs.Such resistance can be mediated by over-expression of genes such asmulti-drug resistance (MDR) pumps and anti-apoptotic genes, amongothers. To combat this resistance, minicells can be packaged withtherapeutically significant concentrations of functional nucleic acid toMDR-associated genes and administered to a patient before chemotherapy.Furthermore, packaging into the same minicell multiple functionalnucleic acid directed to different mRNA targets can enhance therapeuticsuccess since most molecular targets are subject to mutations and havemultiple alleles. More details of directly packaging a nucleic acid intoa minicell is provided in WO 2009/027830, the contents of which areincorporated into the present disclosure in its entirety by reference.

Small molecule drugs, whether hydrophilic or hydrophobic, can bepackaged in minicells by creating a concentration gradient of the drugbetween an extracellular medium containing minicells and the minicellcytoplasm. When the extracellular medium contains a higher drugconcentration than the minicell cytoplasm, the drug naturally moves downthis concentration gradient, into the minicell cytoplasm. When theconcentration gradient is reversed, however, the drug does not move outof the minicells. More details of the drug loading process and itssurprising nature are found, for instance, in U.S. Patent ApplicationPublication No. 2008/0051469.

To load minicells with drugs that normally are not water soluble, thedrugs initially can be dissolved in an appropriate solvent. For example,paclitaxel can be dissolved in a 1:1 blend of ethanol and cremophore EL(polyethoxylated castor oil), followed by a dilution in PBS to achieve asolution of paclitaxel that is partly diluted in aqueous media andcarries minimal amounts of the organic solvent to ensure that the drugremains in solution. Minicells can be incubated in this final medium fordrug loading. Thus, the inventors discovered that even hydrophobic drugscan diffuse into the cytoplasm or the membrane of minicells to achieve ahigh and therapeutically significant cytoplasmic drug load. This isunexpected because the minicell membrane is composed of a hydrophobicphospholipid bilayer, which would be expected to prevent diffusion ofhydrophobic molecules into the cytoplasm.

It is demonstrated the loading into minicells of a diversity ofrepresentative small molecule drugs, illustrating different sizes andchemical properties: Doxorubicin, paclitaxel, fluoro-paclitaxel,cisplatin, vinblastine, monsatrol, thymidylate synthase (TS) inhibitorOSI-7904, irinotecan, 5-fluorouracil, gemcitabine, and carboplatin.Across the board, moreover, the resultant, small molecule drug-packagedminicells show significant anti-tumor efficacy, in vitro and in vivo.These data presented here therefore demonstrate the effectiveness andversatility of the minicell loading methodology.

(G) Directing Minicells or Killed Bacterial Cells to Specific MammalianCells

Pursuant to a further aspect of this disclosure, the minicells or killedbacterial cells of a composition, as described above, are directed to atarget mammalian tumor cell via a ligand. In some embodiments the ligandis “bispecific.” That is, the ligand displays a specificity for bothminicell and mammalian (tumor) cell components, such that it causes agiven vesicle to bind to the target cell, whereby the latter engulfs theformer. Use of bispecific ligands to target a minicell to a tumor cellis further described in WO 05/056749 and WO 05/079854, and use ofbispecific ligands to target a killed bacterial cell to a tumor cell isfurther described in U.S. Pat. No. 8,591,862, the respective contents ofwhich are incorporated here by reference in its entirety. Once such aligand is attached to a vesicle, the unoccupied specificity(“monospecificity”) of the ligand pertains until it interacts with thetarget (tumor) mammalian cell.

The ligand can be attached to the cell membrane of the vesicles byvirtue of the interaction between the ligand and a component on the cellmembrane, such as a polysaccharide, a glycoprotein, or a polypeptide.The expressed ligand is anchored on the surface of a vesicle such thatthe surface component-binding portion of the ligand is exposed so thatthe portion can bind the target mammalian cell surface component whenthe vesicle and the mammalian cell come into contact.

Alternatively, the ligand can be expressed and displayed by a livingcounterpart of a bacterially derived vesicle, e.g., by the parent cellof a minicell or by a bacterial cell before it becomes a killed cell. Inthis instance the ligand does not require a specificity to the vesicleand only displays a specificity to a component that is characteristic ofmammalian cells. That is, such component need not be unique to tumorcells, per se, or even to the particular kind of tumor cells undertreatment, so long as the tumor cells present the component on theirsurface.

Upon intravenous administration, vesicles accumulate rapidly in thetumor microenvironment. This accumulation, occurring as a function ofthe above-described leaky tumor vasculature, effects delivery ofvesicle-packaged therapeutic payload to cells of the tumor, which theninternalize packaged vesicles.

The inventors have found that this delivery approach is applicable to arange of mammalian tumor cells, including cells that normally arerefractory to specific adhesion and endocytosis of minicells. Forinstance, ligands that comprise an antibody directed at an anti-HER2receptor or anti-EGF receptor can bind minicells to the respectivereceptors on a range of targeted non-phagocytic cells, such as lung,ovarian, brain, breast, prostate, and skin cancer cells.

The binding thus achieved precedes uptake of the vesicles by each typeof non-phagocytic cells. That is, in the context of the presentinvention a suitable target cell presents a cell surface component thebinding of which, by a ligand on a vesicle, elicits endocytosis of thatvesicle.

More specifically, the present inventors discovered that the interactionbetween (a) the ligand on a minicell or a killed bacterial cell and (b)a mammalian cell surface receptor can activate an uptake pathway, calledhere a “receptor-mediated endocytosis” (rME) pathway, into thelate-endosomal/lysosomal compartment of the target host cell, such as atumor cell. By this rME pathway, the inventors found, bacteriallyderived vesicles are processed through the early endosome, the lateendosome and the lysosome, resulting in release of their payload intothe cytoplasm of the mammalian host cell. Moreover, a payload that is anucleic acid not only escapes complete degradation in thelate-endosomal/lysosomal compartment but also is expressed by the hostcell.

A ligand for this delivery approach can be “bispecific,” as describedabove, because it binds to surface components on a payload-carryingvesicle and on a target cell, respectively, and its interaction with thelatter component leads to uptake of the vesicle into the rME pathway. Inany event, a given target cell-surface component can be a candidate forbinding by the ligand, pursuant to the invention, if interaction withthe component in effect accesses an endocytic pathway that entails acytosolic internalization from the target cell surface. Such candidatesare readily assessed for suitability in the invention via an assay inwhich a cell type that presents on its surface a candidate component isco-incubated in vitro with minicells carrying a ligand that binds thecandidate and that also is joined to a fluorescent dye or other markeramenable to detection, e.g., visually via confocal microscopy. (An invitro assay of this sort is described by MacDiarmid et al. (2007), inthe legend to FIG. 3 at page 436.) Thus, an observed internalization ofthe marker constitutes a positive indication by such an assay that thetested target cell-surface component is suitable for the presentinvention.

Illustrative of candidate target cell-surface components are members of(A) the receptor tyrosine kinases or “RKTs,” a family of transmembraneproteins that undergo constitutive internalization (endocytosis) at arate similar to that of other integral membrane proteins. See Goh andSorkin, Cold Spring Harb. Perspect. Biol. 5: a017459 (2013). The familyof RKTs is described by Lemmon and Schlessinger, Cell 141(7): 1117-134(2010). The table below lists, in twenty subfamilies, all fifty-eightRTKs in the human proteome, any one or more of which may be tested forsuitability in the invention, as described above (see also FIG. 7).

RTK Subfamilies Exemplary RTKs ErbB EGFR, ErbB2, ErbB3, ErbB4 Ins InsR,IGF1R, InsRR PDGF PDGFRα, PDGFRβ, CSF1R/Fms, Kit/SCFR, Fit3/Flk2 VEGFVEGFR1/Fit1, VEGFR2/KDR, VEGFR3/Fit4 FGF FGFR1, FGFR2, FGFR3, FGFR4 PTK7PTK7/CCK4 Trk TrkA, TrkB, TrkC Ror Ror1, Ror2 MuSK MuSK Met Met, Ron AxlAxl, Mer, Tyro3 Tie Tie1, Tie2 Eph EphA1-8, EphA10, EphB1-4, EphB6 RetRet Ryk Ryk DDR DDR1, DDR2 Ros Ros LMR LMR1, LMR2, LMR3 ALK ALK, LTKSTYK1 SuRTK106/STYK1

Likewise illustrative are members of: (B) the class ofmembrane-associated, high-affinity folate binding proteins (folatereceptor), which bind folate and reduced folic acid derivatives andwhich mediate delivery of tetrahydrofolate to the interior of cells, (C)the subgroup of membrane-bound cytokine receptors that play a role inthe internalization of a cognate cytokine, such as IL13; (D) the surfaceantigens, such as CD20, CD33, mesothelin and HM1.24, that are expressedon certain cancer cells and that mediate the internalization of cognatemonoclonal antibodies, e.g., rituximab in the instance of CD20; and (E)the family of adhesion receptors (integrins), transmembrane glyproteinsthat are trafficked trhough the endosomal pathway and are majormediators of cancer cell adhesion to extracellular matrix.

In accordance with the invention, the ligand can be any polypeptide orpolysaccharide that exhibits the desired specificity or specificities,as the case may be. Preferred ligands are antibodies. In its present usethe term “antibody” encompasses an immunoglobulin molecule obtained byin vitro or in vivo generation of an immunogenic response. Accordingly,the “antibody” category includes monoclonal antibodies and humanizedantibodies, such as single-chain antibody fragments (scFv), bispecificantibodies, etc. A large number of different bispecific protein andantibody-based ligands are known, as evidenced by the review article ofCaravella and Lugovskoy, Curr. Opin. Chem. Biol. 14: 520-28 (2010),which is incorporated here by reference in its entirety. Antibodiesuseful in accordance with the present disclosure can be obtained as wellby known recombinant DNA techniques.

By way of non-limiting example, therefore, an antibody that carriesspecificity for a surface component, such as a tumor antigen, can beused to target minicells to cells in a tumor to be treated, pursuant tothe invention. Illustrative cell surface receptors in this regardinclude any of the RTKs epidermal growth factor receptor (EGFR),vascular endothelial growth factor receptor (VEGFR), platelet-derivedgrowth factor receptor (PDGFR) and insulin-like growth factor receptor(IGFR), each of which is highly expressed in several solid tumors,including brain tumors, and folate receptor, which is overexpressed insome pituitary adenomas. Such a bispecific ligand can be targeted aswell to mutant or variant receptors, e.g., the IL-13Rα2 receptor, whichis expressed in 50% to 80% of human glioblastoma multiforme tumors, seeWykosky et al., Clin Cancer Res. 14: 199-208 (2008), Jarboe et al.,Cancer Res. 67: 7983-86 (2007), Debinski et al., J. Neurooncol. 48:103-11 (2000), and Okada et al., J. Bacteriol. 176: 917-22 (1994), butwhich differs from its physiological counterpart IL4R/IL13R, expressedin normal tissues. See Hershey, J. Allergy Clin. Immunol. 111: 677-90(2003). Thus, IL13Rα2 is virtually absent from normal brain cells. SeeDebinski and Gibo, Mol. Med. 6: 440-49 (2000). Additionally, tumors thatmetastasize to the brain may overexpress certain receptors, which alsocan be suitable targets. For instance, Da Silva et al., Breast CancerRes. 12: R46 (1-13) (2010), showed that brain metastases of breastcancer expressed all members of the HER family of RTKs. HER2 wasamplified and overexpressed in 20% of brain metastases, EGFR wasoverexpressed in 21% of brain metastases, HER3 was overexpressed in 60%of brain metastases and HER4 was overexpressed in 22% of brainmetastases. Interestingly, HER3 expression was increased in breastcancer cells residing in the brain.

(H) Agents to Increase the Levels of IFN-Gamma

The present compositions and methods can further include an agent thatincreases the level (e.g., the activity or expression level) ofIFN-gamma in a patient.

In one embodiment, the agent includes an IFN-gamma protein or analog.Commercial products of IFN-gamma, such as Actimmune®, are or will beavailable. Actimmune® is a bioengineered form of interferon gamma, aprotein that acts as a biologic response modifier through stimulation ofthe human immune system. As noted above, the FDA has approved Actimmune®for use in children and adults with chronic granulomatous disease andsevere, malignant osteopetrosis.

IFN-gamma production is controlled by cytokines secreted by APCs, mostnotably interleukin (IL)-12 and IL-18. These cytokines serve as a bridgeto link infection with IFN-gamma production in the innate immuneresponse. Macrophage recognition of many pathogens induces secretion ofIL-12 and chemokines. These chemokines attract NK cells to the site ofinflammation, and IL-12 promotes IFN-gamma synthesis in these cells. Inmacrophages, NK and T cells, the combination of IL-12 and IL-18stimulation further increases IFN-gamma production. Accordingly, any ofthese proteins or their combinations are suitable agents for the purposeof this disclosure.

Negative regulators of IFN-gamma production include IL-4, IL-10,transforming growth factor-β, and glucocorticoids. Proteins or nucleicacids that inhibit these factors will be able to stimulate theproduction of IFN-gamma.

Also suitable for use in this context are polynucleotides that encodeIFN-gamma or genes that activate the production and/or the secretion ofIFN-gamma.

The agent that increases the level of IFN-gamma also can be a viralvaccine. A number of viral vaccines are available that can induceIFN-gamma production without causing infection or other types of adverseeffects. Illustrative of this class of viral-vaccine agent is a flu(influenza) vaccine.

The data show that the serum concentration of IFN-gamma required foreffectively activating host immune response to tumor cells is low, whenthe patient also receives administration of drug-loaded, bispecificantibody-targeted minicells or killed bacterial cells. Thus, in oneaspect the inventive methodology results in increase of serum IFN-gammaconcentration that is not higher than about 30,000 pg/mL. In anotheraspect, the serum IFN-gamma concentration is increased to not higherthan about 5000 pg/mL, 1000 pg/mL, 900 pg/mL, 800 pg/mL, 700 pg/mL, 600pg/mL, 500 pg/mL, 400 pg/mL, 300 pg/mL, 200 pg/mL, or 100 pg/mL. In afurther aspect, the resulting serum IFN-gamma concentration is at leastabout 10 pg/mL, or at least about 20 pg/mL, 30 pg/mL, 40 pg/mL, 50pg/mL, 60 pg/mL, 70 pg/mL, 80 pg/mL, 90 pg/mL, 100 pg/mL, 150 pg/mL, 200pg/mL, 300 pg/mL, 400 pg/mL or 500 pg/mL.

Pursuant to some aspects, the agent is an IFN-gamma protein, engineeredprotein or analog. In some aspects, the administration achieves fromabout 0.02 ng to 1 microgram of IFN-gamma per ml of host blood. In oneaspect, the achieved IFN-gamma concentration in the host blood is fromabout 0.1 ng to about 500 ng per ml, or from about 0.2 ng to about 200ng per ml, or from about 0.5 ng to about 100 ng per ml, or from about 1ng to about 50 ng per ml, or from about 2 ng to about 20 ng per ml. Thetherapeutic dose of IFN-gamma in the composition of the presentdisclosure can be determined accordingly.

(I) Formulations and Administration Routes and Schedules

Formulations of a composition of the disclosure can be presented in unitdosage form, e.g., in ampules or vials, or in multi-dose containers,with or without an added preservative. The formulation can be asolution, a suspension, or an emulsion in oily or aqueous vehicles, andcan contain formulatory agents, such as suspending, stabilizing and/ordispersing agents. A suitable solution is isotonic with the blood of therecipient and is illustrated by saline, Ringer's solution, and dextrosesolution. Alternatively, formulations can be in lyophilized powder form,for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-freewater or physiological saline. The formulations also can be in the formof a depot preparation. Such long-acting formulations can beadministered by implantation (for instance, subcutaneously orintramuscularly) or by intramuscular injection.

In some aspects, a minicell- or killed bacterial cell-containingcomposition that includes a therapeutically effective amount of ananti-neoplastic agent is provided. A “therapeutically effective” amountof an anti-neoplastic agent is a dosage of the agent in question, e.g.,a siRNA or a chemotherapeutic drug that invokes a pharmacologicalresponse when administered to a subject, in accordance with the presentdisclosure.

In some aspects, a composition is provided that includes atherapeutically effective amount of an agent that increases the level ofIFN-gamma. In some aspects, a composition, kit, package or product isprovide that includes both a minicell or killed bacterial cell asdescribed and an agent that increases the level of IFN-gamma.

In the context of the present disclosure, therefore, a therapeuticallyeffective amount can be gauged by reference to the prevention oramelioration of the tumor or a symptom of tumor, either in an animalmodel or in a human subject, when minicells or killed bacterial cellscarrying a therapeutic payload are administered, as further describedbelow. An amount that proves “therapeutically effective amount” in agiven instance, for a particular subject, may not be effective for 100%of subjects similarly treated for the tumor, even though such dosage isdeemed a “therapeutically effective amount” by skilled practitioners.The appropriate dosage in this regard also will vary as a function, forexample, of the type, stage, and severity of the tumor. Likewise, when“therapeutically effective” is used to refer to the number of minicellsin a pharmaceutical composition, the number can be ascertained based onwhat anti-neoplastic agent is packaged into the minicells and theefficacy of that agent in treating a tumor. The therapeutic effect, inthis regard, can be measured with a clinical or pathological parametersuch as tumor mass. A reduction or reduced increase of tumor mass,accordingly, can be used to measure therapeutic effects.

With respect to the agent that increases the level of IFN-gamma, a“therapeutically effective amount” can refer to the amount of the agent,upon administration, achieves the desired host blood concentration, asprovided supra.

Formulations within the disclosure can be administered via variousroutes and to various sites in a mammalian body, to achieve thetherapeutic effect(s) desired, either locally or systemically. In aparticular aspect, the route of administration is intravenous injection.

In general, formulations of the disclosure can be used at appropriatedosages defined by routine testing, to obtain optimal physiologicaleffect, while minimizing any potential toxicity. The dosage regimen canbe selected in accordance with a variety of factors including age,weight, sex, medical condition of the patient; the severity or stage oftumor, the route of administration, and the renal and hepatic functionof the patient.

Optimal precision in achieving concentrations of minicell, killedbacterial cell, and therapeutic agent within the range that yieldsmaximum efficacy with minimal side effects can and typically willrequire a regimen based on the kinetics of agent availability to targetsites and target cells. Distribution, equilibrium, and elimination ofminicells or agent can be considered when determining the optimalconcentration for a treatment regimen. The dosage of minicells andtherapeutic agent, respectively, can be adjusted to achieve desiredeffects.

Moreover, the dosage administration of the formulations can be optimizedusing a pharmacokinetic/pharmacodynamic modeling system. Thus, one ormore dosage regimens can be chosen and a pharmacokinetic/pharmacodynamicmodel can be used to determine the pharmacokinetic/pharmacodynamicprofile of one or more dosage regimens. Based on a particular suchprofile, one of the dosage regimens for administration then can beselected that achieves the desired pharmacokinetic/pharmacodynamicresponse. For example, see WO 00/67776.

The minicells or killed bacterial cells packaged with an anti-neoplasticagent and the agent that increases the level of IFN-gamma can beadministered concurrently, either in a combination formulation or asseparate compositions, or sequentially one after the other. Whenadministered sequentially, the minicells or killed bacterial cells canbe administered before the agent that increases the level of IFN-gamma,or afterwards. In one aspect, when the minicells or killed bacterialcells reach maximum plasma level or effective plasma level followingadministration, the host has achieved or is maintaining a minimum levelof IFN-gamma. Such a minimum level is one that is required to producesynergism between the compositions. This can be achieved byadministering the agent that increases the IFN-gamma level beforeadministering the minicells or killed bacterial cells, or byadministering the agent shortly after the minicells or killed bacterialcells are administered, in particular at a relatively high dose. It isnoted that administration of both compositions can take place in series.In that respect, then, the administrations result in constant exposureof the host to both the minicells or killed bacterial cells and theagent that increases IFN-gamma.

A formulation or combination of formulations of the disclosure can beadministered at least once a week to a tumor patient, over the course ofseveral weeks. Thus, the formulation can be administered at least once aweek, over a period of several weeks to several months.

More specifically, inventive formulations can be administered at leastonce a day for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days.Alternatively, the formulations can be administered about once every dayor about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days ormore.

In another embodiment of the disclosure, formulations can beadministered about once every week or about once every 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks or more.Alternatively, the formulations can be administered at least once a weekfor about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20 weeks or more.

Alternatively, the formulations can be administered about once everymonth or about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months ormore.

The formulations can be administered in a single daily dose.Alternatively, the total daily dosage can be administered in divideddoses of two, three, or four times daily.

The following examples are illustrative only, rather than limiting, andprovide a more complete understanding of the disclosure.

Example 1. Tumor Size Reduction Correlated with Interferon-Gamma Levels

This example demonstrates that the reduction of brain tumor volumes indogs undergoing treatments with drug-loaded minicells correlated withthe expression level of interferon-gamma (IFNγ). This example,therefore, suggests that IFN-gamma increases the efficacy of drug-loadedminicells. Given the low amount of IFN-gamma needed, this examplefurther suggests synergism between IFN-gamma and drug-loaded, bispecificantibody-targeted minicells.

Materials and Methods Preparation and Dosing of Doxorubicin-Packaged,Canine EGFR-Targeted Minicells

Minicells were derived from a minCDE-chromosomal deletion mutant ofSalmonella enterica serovar Typhimurium, S. typhimurium, purified,packaged with doxorubicin (dox) and targeted via attachment of abispecific monoclonal antibody (MAb) comprising anti-minicell surfaceO-polysaccharide and anti-canine EGFR specificities, (designated,^(EGFR)minicells_(Dox)).

Dogs in this study were pet dogs presenting as patients to theVeterinary Specialist Centre (VSC) or the Small Animal SpecialistHospital (SASH), in Sydney, Australia. Study participation was offeredto patients where standard therapy had been declined by the dog's owneror, in cases of advanced disease, where no meaningful standard therapyexisted. Dogs were treated in compliance with guidelines promulgated bythe National Health and Medical Research Council (Australia) for thecare and use of laboratory animals, and with EnGeneIC Animal EthicsCommittee approval. Signed informed consent was obtained from allowners.

All brain tumors were diagnosed by histology or cytology where feasible.Antemortem diagnoses were based on a combination of characteristicappearance on magnetic resonance imaging (MRI) and clinical signs.Histological diagnosis was deemed too invasive in these brain tumorcases and diagnosis was confirmed by necropsy.

Treatment with 1×10^(10 EGFR)minicells_(Dox) per dose was performed on aweekly basis. Treatment was administered via an aseptically placedperipheral vein catheter (left cephalic) in 2 ml over a 2 minuteinfusion.

MRI Tumor Imaging

Tumor images were performed at Specialist Magnetic Resonance Imagingusing a Philips 1.5T Achieva scanner. The protocol used an 8-channelhead coil or 8-channel knee coil depending on the size of the dog (smalldogs used the knee coil).

Sequences were obtained from sagittal T1, axial T2, Coronal GradientEcho, axial diffusion weighted images (DWI) pre contrast, coronalvolumetric fluid-attenuated inversion recovery (FLAIR) and postgadolinium T1 weighted images obtained in three planes.

IFN-Gamma Enzyme-Linked Immunosorbent Assay (ELISA)

Blood was taken before minicell dose and serum received directly fromthe veterinary clinic. IFN-gamma measurement performed in duplicateusing Canine IFN-gamma DuoSet ELISA Kit from Development System(#DY781B) as per the manufacturer's instructions.

Results

The results, in terms of tumor volume changes, in three dogs receivingthe minicell treatments are presented in FIG. 1A-1C. The line graphsrepresent tumor measurements (lefty axis=volume in (mm³) or longestdiameter (mm)) as a function of the number of minicells doses (x axis).Crosses represent the doses at which the tumors were visualized andmeasured by magnetic resonance imaging (MM).

During the treatment, two of the dogs showed drastic reduction in tumorvolumes in certain treatment periods (dogs A and B in FIGS. 1A and 1B).It was discovered unexpectedly that the dogs were suffering from viralinfections during those periods. Among a number of parameters examinedthat might be associated with viral infection, it was found that theserum concentration of IFN-gamma highly correlated with the tumor volumereduction rates.

In each of FIG. 1A-1C, the triangular markers represent serum interferongamma (IFNγ) levels; measured at the indicated doses by ELISA. Theright-handy axis indicates IFN-gamma levels in pg/mL. Where the assaywas performed but IFN-gamma was below the detection limit of the assay(<56 pg/mL), the data points are represented by triangular markers at 0pg/mL. Where tumor length (1), width (w) and height (h) measurementswere made, the tumor volume (V) was calculated using the ellipsoidformula (V=(π/6)*l*w*h).

These figures thus demonstrate a strong correlation between serumIFN-gamma levels and brain tumor volume reduction rates. What also issurprising is that any detectable IFN-gamma levels led to increasedanti-tumor response. The lowest IFN-gamma level directed was about 500pg/mL, at dose 41 in dog B (FIG. 1B). Such a drastic effect of IFN-gammaon the tumor treatment of drug-loaded minicells is a strong indicationof synergism between them.

Example 2. Significant Tumor Regression in Mouse Xenografts (HumanAlveolar Adenocarcinoma) Following Treatment with ^(EGFR)Minicells_(Dox)and IFN-Gamma

This example demonstrates that combined treatment with bispecificligand-targeted and doxorubicin-packaged intact minicells with IFN-gammacan effect regression of human alveolar adenocarcinoma tumor xenograftsestablished in 6 week-old female athymic nude mice.

As described above, minicells were produced from an S. typhimuriumminCDE-mutant strain and were purified using a gradientcentrifugation/filamentation/filtration/endotoxin removal procedurepreviously described in MacDiarmid et al. (2007). The purified minicellswere packaged with chemotherapeutic drug doxorubicin, also perMacDiarmid et al. (2007).

The bispecific antibody (BsAb) was a single polypeptide containingbinding specificity respectively for S. typhimurium O-polysaccharide,present on minicells, and for human EGFR, overexpressed on alveolaradenocarcinoma cells. The O-polysaccharide specificity was derived froma mouse monoclonal antibody, for which the variable regions wereisolated from a hybridoma cell line and presented as a single-chainvariable fragment (scFv). The hybridoma cell line was prepared byimmunizing mice with purified LPS and fusing the lymphocytes with tumorcells. Subsequently, the clones were screened for an antibody capable ofbinding the O-polysaccharide. The EGFR specificity, also presented as anscFv, was derived from the commercial antibody Erbitux® (Bristol MyersSquibb, USA). The two scFv components were separated by a flexiblelinker and a 6×His tag incorporated at the N-terminus, to facilitatepurification by immobilized metal affinity chromatography, and a c-myctag at the C-terminus, to aid in additional detection. Linkersconnecting the scFv components are well known, as evidenced by Gall etal., Protein Engineering, Design and Selection 17: 357-66 (2004), forexample.

The expression vector encoding the BsAb contains an hCMV promoter forhigh-level expression and a signal peptide for the secretion of the BsAbinto the cell culture medium. The expression vector encoding the BsAb isstably transfected into suspension adapted Chinese hamster ovary (CHO)cells in chemically defined, protein and animal origin free medium andthe protein is expressed over 10 days in culture.

Two chromatographic columns were used to purify the antibody: animmobilized metal ion affinity chromatography column (IMAC-HisTrapExcel) and a hydroxyapatite chromatography column (BioRad CHT I). Thisapproach achieved an antibody purity of >98%. For viral safety ofproduct, the antibody was put through a solvent/detergent inactivation,using TNBP/Tween, and a viral filtration. The final yield of antibodywas 10 mg from 1 L of cell culture supernatant.

The mice used in this example were purchased from Animal ResourcesCentre (Perth, Australia), and all animal experiments were performed incompliance with GUIDE OF CARE AND USE OF LABORATORY ANIMALS, 8^(th) ed.(National Academies Press, 2011) and with Animal Ethics Committeeapproval. The experiments were performed in the NSWAgriculture-accredited small animal facility at EnGeneIC Ltd. (Sydney,Australia).

Human alveolar adenocarcinoma cells (A549, ATCC) were grown in tissueculture to full confluency in T-75 flasks in GIBCO®-RPMI 1640 medium, aproduct of Life Technologies (Carlsbad, Calif., USA), supplemented with5% bovine calf serum and glutamine, in a humidified atmosphere of 95%air and 5% CO₂ at 37° C. Cells (1×10⁶) in 50 μL serum-free medium with50 μL growth factor reduced Matrigel®, product of BD Biosciences(Franklin Lakes, N.J.). The cells then were injected subcutaneously,using a 23-gauge needle, between the shoulder blades of each mouse.

The resulting tumors were measured twice a week, using an electronicdigital caliper (Mitutoyo, Japan, with a measure accuracy of +/−0.001inch), and mean tumor volume was calculated using the formula

length (mm)×width (mm²)×0.5=volume (mm³).

The treatments commenced when the tumors reached a mean of ˜285 mm³, andmice were randomized to four different groups of seven mice per group.All treatments were administered intravenously (i.v.) in a total volumeof 100 μl. All minicell doses contained 1×10⁹ minicells of therespective type.

In terms of experimental design, Group 1 (control) received no sterilephysiological saline. Group 2 (control) received IFN-gamma (0.5×10⁴ IU)per dose. Group 3 (control) received ^(EGFR)minicells_(Dox). Group 4(experimental) received ^(EGFR)minicells_(Dox) and IFN-gamma (0.5×10⁴IU) per dose.

The results (FIG. 2) revealed that mice treated with^(EGFR)minicells_(Dox) (Group 3) achieved tumor stabilization. Bycontrast, mice treated with ^(EGFR)minicells_(Dox) and IFN-gamma (Group4) showed highly significant tumor regression by day 43, after a totalof six doses. Mice treated with IFN-gamma alone (Group 2) showed noanti-tumor efficacy, and the tumors grew as in the saline treated group(Group 1).

Example 3. Significant Tumor Regression in Mouse Xenografts (HumanBreast Cancer—Moderate Sized Tumors ˜145 mm³) after Treatment with^(EGFR)Minicells_(Dox) and IFN-Gamma

This example demonstrates that combined treatment with bispecificligand-targeted and doxorubicin-packaged intact minicells with IFN-gammacan effect regression of human breast tumor xenografts established in 6week-old female athymic nude mice.

As described above, minicells were purified, packaged with doxorubicin,and targeted using single chain bispecific antibody withanti-O-polysaccharide and anti-EGFR specificities. Additionally, humanbreast cancer cells (MDA-MB-468; ATCC) were established as xenografts innu/nu mice, and tumor volumes were measured, also as described.

The treatments commenced when the tumors reached a mean of ˜145 mm³, andmice were randomized to four different groups of seven mice per group.All treatments were administered i.v. in a total volume of 100 μl. Allminicell doses contained 1×10⁹ minicells of the respective type.

The experiment was designed as follows: Group 1 (control) receivedsterile physiological saline only. Group 2 (control) received IFN-gamma(0.5×10⁴ IU) per dose. Group 3 (control) received^(EGFR)minicells_(Dox). Group 4 (experimental) received^(EGFR)minicells_(Dox) and IFN-gamma (0.5×10⁴ IU) per dose.

The results as shown in FIG. 3 revealed that mice treated with^(EGFR)minicells_(Dox) (Group 3) achieved tumor stabilization, but byabout day 25 the tumors began to grow again, probably due to developmentof resistance to doxorubicin. By contrast, mice treated with^(EGFR)minicells_(Dox) and IFN-gamma (Group 4) showed highly significanttumor regression, and by day 30 these tumors, after a total of sixdoses, were more like scar tissue. Mice treated with IFN-gamma alone(Group 2) showed no anti-tumor efficacy, and the tumors grew as in thesaline treated group (Group 1).

Example 4. Significant Tumor Regression in Mouse Xenografts (HumanBreast Cancer—Large Tumors ˜250 mm³) after Treatment with^(EGFR)Minicells_(Dox) and IFN-Gamma

This example demonstrates that combined treatment of with bispecificligand-targeted and doxorubicin-packaged intact minicells with IFN-gammacan effect regression even in large sized tumors (˜250 mm³) of humanbreast tumor xenografts established in 6 week-old female athymic nudemice.

As described above, minicells were purified, packaged with doxorubicinand targeted using single chain bispecific antibody withanti-O-polysaccharide and anti-EGFR specificities. Human breast cancercells (MDA-MB-468) were established as xenografts in nu/nu mice, andtumor volumes were measured, also as described above.

The treatments were begun when the tumors reached a mean of ˜250 mm³. Asabove, mice were randomized to four different groups of seven mice pergroup. The i.v. administration and minicell doses were as above, too.

The experiment was designed as follows. Group 1 (control) receivedsterile physiological saline only. Group 2 (control) received IFN-gamma(0.5×10⁴ IU) per dose. Group 3 (control) received^(EGFR)minicells_(Dox). Group 4 (experimental) received^(EGFR)minicells_(Dox) and IFN-gamma (0.5×10⁴ IU) per dose.

The results as shown in FIG. 4 revealed that mice treated with^(EGFR)minicells_(Dox) (Group 3) achieved tumor regression but that by˜day 23 the tumors had begun to grow again; as before, development ofdoxorubicin resistance was the likely cause. On the other hand, micetreated with ^(EGFR)minicells_(Dox) and IFN-gamma (Group 4) showedhighly significant tumor regression, and after a total of 3 doses (i.e.,by day 28) these tumors were more like scar tissue. Mice treated withIFN-gamma alone (Group 2) showed no anti-tumor efficacy and the tumorsgrew as in the saline treated group (Group 1).

Example 5. Tumor Regression in Mouse Xenografts (Human BreastCancer—Very Large Tumors ˜250 mm³ to 600 mm³) after Treatment with^(EGFR)Minicells_(Dox) and IFN-Gamma

This example demonstrates that combined treatment with bispecificligand-targeted and doxorubicin-packaged intact minicells with IFN-gammacan effect regression even in very large sized tumors (˜250 mm³ to 600mm³) of human breast tumor xenografts established in 6 week old femaleathymic nude mice.

As described above, minicells were purified, packaged with doxorubicinand targeted using single chain bispecific antibody withanti-O-polysaccharide and anti-EGFR specificities. Also as described,human breast cancer (MDA-MB-468) cells were established as xenografts innu/nu mice, and tumor volumes were measured.

The treatments were commenced when the tumors reached ˜250 mm³ to 600mm³. Individual mice were treated with ^(EGFR)minicells_(Dox) andIFN-gamma (0.5×10⁴ IU) per dose. All treatments were administered i.v.in a total volume of 100 μl, and all minicell doses contained 1×10⁹minicells of the respective type.

The results are depicted in FIG. 5. Notwithstanding the large size ofthe tumors, all four mice achieved tumor regression. This shows thateven very large tumors (˜600 mm³), where mice would normally beeuthanized, can be treated effectively with the combination of^(EGFR)minicells_(Dox) and IFN-gamma (0.5×10⁴ IU).

Example 6. Significant Tumor Regression in Mouse Xenografts (HumanAlveolar Adenocarcinoma) after Treatment with ^(EGFR)Minicells_(Dox) andTwo Dose Levels of IFN-Gamma

This example demonstrates that combined treatment with bispecificligand-targeted and doxorubicin-packaged intact minicells with IFN-gammaat two different dose levels can effect regression of human alveolaradenocarcinoma xenografts established in 6 week-old female athymic nudemice.

As described above, minicells were purified, packaged with doxorubicinand targeted using single chain bispecific antibody withanti-O-polysaccharide and anti-EGFR specificities. Human alveolaradenocarcinoma (A549) cells were established as xenografts in nu/numice, and tumor volumes were measured, also as described above.

The treatments commenced when the tumors reached a mean of ˜100 mm³, andmice were randomized to four different groups of seven mice per group.All treatments were administered i.v. in a total volume of 100 μl. Allminicell doses contained 1×10⁹ minicells of the respective type.

Group 1 (control) received no sterile physiological saline. Group 2(control) received ^(EGFR)minicells_(Dox) (twice per week). Group 3(experimental) received ^(EGFR)minicells_(Dox) and IFN-gamma (0.75×10⁴IU) per dose, twice per week. Group 4 (experimental) received^(EGFR)minicells_(Dox) and IFN-gamma (0.5×10⁴ IU) per dose, three perweek.

As FIG. 6 shows, mice treated with ^(EGFR)minicells_(Dox) and IFN-gammaat both doses (0.5×10⁴ IU and 0.75×10⁴ IU; Groups 3 and 4) achievedtumor stabilization. By contrast, mice treated with^(EGFR)minicells_(Dox) (Group 2) showed no anti-tumor efficacy, and thetumors grew as in the saline treated group (Group 1). These datademonstrate that combining IFN-gamma with ^(EGFR)minicells_(Dox) wasessential at both IFN-gamma dose levels to achieve tumor stabilizationin the treatment of tumors that normally are resistant to eitherIFN-gamma treatment alone or ^(EGFR)minicells_(Dox) treatment alone.

1-14. (canceled)
 15. A package, product, or kit comprising: (a) a firstcomposition comprising a plurality of bacterially derived intactminicells or intact killed bacterial cells, each of which encompasses ananti-neoplastic agent and carries a ligand on the surface of the intactminicells or intact killed bacterial cells, wherein the ligand hasspecificity to a non-phagocytic mammalian cell surface receptor, and (b)a second composition comprising interferon-gamma (IFN-gamma) or an agentthat increases the expression of IFN-gamma in the subject.
 16. Acomposition comprising: (a) a plurality of bacterially derived intactminicells or intact killed bacterial cells, each of which encompasses ananti-neoplastic agent and carries a ligand on the surface of the intactminicells or bacterial cells, wherein the ligand has specificity to anon-phagocytic mammalian cell surface receptor, and (b) interferon-gamma(IFN-gamma) or an agent that increases the expression of IFN-gamma in asubject upon administration to the subject.
 17. A composition comprisinga plurality of bacterially derived intact minicells or intact killedbacterial cells, each of which encompasses an anti-neoplastic agent andcarries a ligand on the surface of the intact minicells or intact killedbacterial cells, wherein the ligand has specificity to a non-phagocyticmammalian cell surface receptor, wherein: (a) the anti-neoplastic agentis a functional nucleic acid or a polynucleotide encoding a functionalnucleic acid; and (b) the functional nucleic acid inhibits a gene thatpromotes resistance to chemotherapy, wherein the gene is RRM1.
 18. Thepackage, product, or kit of claim 15, wherein the antineoplastic agentis selected from the group consisting of: a radionuclide, a chemotherapydrug, a functional nucleic acid selected from the group consisting ofsiRNA, miRNA, shRNA, lincRNA, antisense RNA, and ribozyme, and apolynucleotide from which a functional nucleic acid selected from thegroup consisting of siRNA, miRNA, shRNA, lincRNA, antisense RNA, andribozyme is transcribed.
 19. The package, product, or kit of claim 15,wherein the agent that increases the expression of IFN-gamma is a viralvaccine that induces IFN-gamma expression or a polynucleotide encodingIFN-gamma.
 20. The package, product, or kit of claim 15, wherein thesecond composition comprises purified IFN-gamma protein.
 21. Thepackage, product, or kit of claim 15, wherein the anti-neoplastic agentis a radionuclide.
 22. The package, product, or kit of claim 15, whereinthe anti-neoplastic agent is a chemotherapy drug.
 23. The package,product, or kit of claim 22, wherein the chemotherapy drug is a smallmolecule drug having a molecular weight of less than about 900 Dalton.24. The package, product, or kit of claim 23, wherein the small moleculedrug is cytotoxic.
 25. The package, product, or kit of claim 23, whereinthe small molecule drug is a morpholinyl anthracycline derivative. 26.The package, product, or kit of claim 15, wherein the anti-neoplasticagent is a functional nucleic acid selected from the group consisting ofsiRNA, miRNA, shRNA, lincRNA, antisense RNA, ribozyme, and apolynucleotide from which a functional nucleic acid selected from thegroup consisting of siRNA, miRNA, shRNA, lincRNA, antisense RNA, andribozyme is transcribed.
 27. The package, product, or kit of claim 26,wherein the functional nucleic acid inhibits a gene that promotes tumorcell proliferation, angiogenesis or resistance to chemotherapy and/orthat inhibits apoptosis or cell cycle arrest.
 28. The package, product,or kit of claim 26, wherein the functional nucleic acid is siRNA ormiRNA.
 29. The package, product, or kit of claim 26, wherein thefunctional nucleic acid inhibits the gene ribonucleotide reductase M1(RRM1).
 30. The package, product, or kit of claim 15, wherein thecomposition comprises from about 10⁹ to about 10¹⁰ minicells or killedbacterial cells.
 31. The composition of claim 16, wherein theantineoplastic agent is selected from the group consisting of aradionuclide, a chemotherapy drug, a functional nucleic acid selectedfrom the group consisting of siRNA, miRNA, shRNA, lincRNA, antisenseRNA, and ribozyme, and a polynucleotide from which a functional nucleicacid selected from the group consisting of siRNA, miRNA, shRNA, lincRNA,antisense RNA, and ribozyme is transcribed.
 32. The composition of claim16, wherein the agent that increases the expression of IFN-gamma is aviral vaccine that induces IFN-gamma expression or a polynucleotideencoding IFN-gamma.
 33. The composition of claim 16, wherein theinterferon-gamma (IFN-gamma) comprises purified IFN-gamma protein. 34.The composition of claim 16, wherein the anti-neoplastic agent is aradionuclide.
 35. The composition of claim 16, wherein theanti-neoplastic agent is a chemotherapy drug.
 36. The composition ofclaim 35, wherein the chemotherapy drug is a small molecule drug havinga molecular weight of less than about 900 Dalton.
 37. The composition ofclaim 36, wherein the small molecule drug is cytotoxic.
 38. Thecomposition of claim 36, wherein the small molecule drug is amorpholinyl anthracycline derivative.
 39. The composition of claim 16,wherein the anti-neoplastic agent is a functional nucleic acid selectedfrom the group consisting of siRNA, miRNA, shRNA, lincRNA, antisenseRNA, ribozyme, and a polynucleotide from which a functional nucleic acidselected from the group consisting of siRNA, miRNA, shRNA, lincRNA,antisense RNA, and ribozyme is transcribed.
 40. The composition of claim39, wherein the functional nucleic acid inhibits a gene that promotestumor cell proliferation, angiogenesis or resistance to chemotherapyand/or that inhibits apoptosis or cell cycle arrest.
 41. The compositionof claim 39, wherein the functional nucleic acid is siRNA or miRNA. 42.The composition of claim 16, wherein the composition comprises fromabout 10⁹ to about 10¹⁰ minicells or killed bacterial cells.